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Ultrafiltration Application and Product Guide

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Application and Product Guide

Lit. No. TP0040EN00 Rev. B 10/07 Printed in U.S.A. 07-493

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Table of Contents
Overview of Membrane Filtration
Membrane Processes . . . . . . . . . . . . . . . . . 4
Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Mode of Operation . . . . . . . . . . . . . . . . . . 9
Diafiltration . . . . . . . . . . . . . . . . . . . . . . . . 10
Fractionation . . . . . . . . . . . . . . . . . . . . . . . 11
Membranes and Devices for Ultrafiltration
Membrane Selection . . . . . . . . . . . . . . . . . . 14
Device Selection . . . . . . . . . . . . . . . . . . . . 16
Microcon Centrifugal Filters . . . . . . . . . . . . . 17
Amicon Ultra-4 Centrifugal Filters . . . . . . . . . 18
Amicon Ultra-15 Centrifugal Filters . . . . . . . . . 20
Centriprep Centrifugal Filters . . . . . . . . . . . . 22
Centricon Plus-70 Centrifugal Filters . . . . . . . . 23
MultiScreen Filter Plate . . . . . . . . . . . . . . . . 24
Ultrafree Centrifugal Filters . . . . . . . . . . . . . . 25
Pellicon XL Cassettes and
Labscale TFF System . . . . . . . . . . . . . . . . . 26
Pellicon 2 Mini Cassettes . . . . . . . . . . . . . . . 28
Prep/Scale Spiral Wound Filter Cartridges . . . 29
Stirred Cells . . . . . . . . . . . . . . . . . . . . . . . . 30
Ultrafiltration Discs . . . . . . . . . . . . . . . . . . . . 31
Protocols for Proteins
Concentration, Desalting and Buffer Exchange . 34
Detergent Removal . . . . . . . . . . . . . . . . . . . 38
Two-Dimensional Electrophoresis
Sample Preparation . . . . . . . . . . . . . . . . . . . 40
Purification of Serum Peptides for Biomarker . . . 42
Rapid Antibody Concentration . . . . . . . . . . . . 45
Affinity Purification . . . . . . . . . . . . . . . . . . . . 46
Removal of Unincorporated Label
from Labeled Protein . . . . . . . . . . . . . . . . . . 50
Rapid Purification of Monoclonal Antibodies . . 52
A Simple Strategy for Protein Fractionation . . . . 54
Urine Concentration . . . . . . . . . . . . . . . . . . 56
Use of Centrifugal Filter Devices as an
Alternative to Stirred Cells . . . . . . . . . . . . . . . 58
Protocols for Nucleic Acids
Purification of DNA Sequencing Reactions . . . 60
Concentrating and Desalting DNA or RNA . . . 63
Preparing Samples for Forensics
Identification Analysis . . . . . . . . . . . . . . . . . 66
Concentration of Genomic DNA for
Forensic Analysis . . . . . . . . . . . . . . . . . . . . 67
Purification of PCR Products . . . . . . . . . . . . . . 68
Quantitative Recoveries of Nanogram
Amounts of Nucleic Acids . . . . . . . . . . . . . . 70
RNA Purification and Preparation of
Fluorescent cDNA Probe from Human mRNA . . 72
Purification of In Vitro Synthesized mRNA . . . . . 76
Effect of Centrifugal Ultrafiltration on
Large Fragment DNA Integrity . . . . . . . . . . . . 78
DNA Extraction from Agarose Gels . . . . . . . . 80
PCR Purification . . . . . . . . . . . . . . . . . . . . . 82
Enzyme Removal . . . . . . . . . . . . . . . . . . . . 84
Protocols for Virus concentration
Concentration of Bacteriophage Using
Ta bl e o f C o n t e n t s
Ultrafiltration . . . . . . . . . . . . . . . . . . . . . . . 88
Concentration of Animal Viruses Using
Ultrafiltration . . . . . . . . . . . . . . . . . . . . . . . 91
Appendix
High Throughput Applications . . . . . . . . . . . . 94
Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . 95
1
2
Ta bl e o f C o n t e n t s
Overview of Membrane Processes . . . . . . . . . . . . . . . . . . . 4

Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Membrane Filtration Mode of Operation . . . . . . . . . . . . . . . . . . . . 9

Diafiltration . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Fractionation . . . . . . . . . . . . . . . . . . . . . . . . . 11

O v e r v ie w o f M e m br a ne F ilt r at i o n

3
 Membrane Processes
Ultrafiltration Ultrafiltration is typically used to separate proteins
from buffer components for buffer exchange,
Ultrafiltration (UF) is the process of separating
desalting, or concentration. Ultrafilters are also ideal
extremely small particles and dissolved molecules
for removal or exchange of sugars, non-aqueous
from fluids. The primary basis for separation is
solvents, the separation of free from protein-bound
molecular size, although in all filtration applications,
ligands, the removal of materials of low molecular
the permeability of a filter medium can be affected
weight, or the rapid change of ionic and/or pH
by the chemical, molecular or electrostatic proper-
environment (see Figure 5, page 10). Depending on
ties of the sample. Ultrafiltration can only separate
the protein to be retained, the most frequently used
molecules which differ by at least an order of
membranes have a nominal molecular weight limit
magnitude in size. Molecules of similar size can
(NMWL) of 3 kDa to 100 kDa.
not be separated by ultrafiltration (see Figure 1).
Ultrafiltration is far gentler to solutes than
Materials ranging in size from 1K to 1000K
processes such as precipitation. UF is more efficient
molecular weight (MW) are retained by certain
because it can simultaneously concentrate and
ultrafiltration membranes, while salts and water will
desalt solutes. It does not require a phase change,
pass through. Colloidal and particulate matter can
which often denatures labile species, and UF can
also be retained. Ultrafiltration membranes can be
be performed either at room temperature or in a
used both to purify material passing through the
cold room.
filter and also to collect material retained by the
filter. Materials significantly smaller than the pore
Microfiltration
size rating pass through the filter and can be
Microfiltration (MF) is the process of removing
depyrogenated, clarified and separated from high
particles or biological entities in the 0.025 m to
O v e r v ie w o f M e m br a ne F ilt r at i o n M e m b r a n e P ro ce s s e s

molecular weight contaminants. Materials larger

10.0 m range from fluids by passage through a
than the pore size rating are retained by the filter
microporous medium such as a membrane filter.
and can be concentrated or separated from low
Although micron-sized particles can be removed
molecular weight contaminants.

Figure 1. Comparison of ultrafiltration with other commonly used membrane separation techniques

Reverse Osmosis Ultrafiltration Microfiltration Clarification

0.0001 m 0.001 m 0.01 m 0.1 m 1 m 10 m 100 m

0.2 kDa 200 kDa 20,000 kDa

Red Blood Smallest

Sugars Proteins B. diminut a
Cell Visible Particle

Amino Acids
Carbon Black Yeast Pollens
Nucleotides

Oligo- Polio
Salts Mycoplasma E. coli Clouds Fog
nucleotides Virus

Human
Antibiotics Mammalian Virus Bacteria
Hair

4
by use of non-membrane or depth materials such as Reverse Osmosis
those found in fibrous media, only a membrane filter
Reverse osmosis (RO) separates salts and small
having a precisely defined pore size can ensure
molecules from low molecular weight solutes
quantitative retention. Membrane filters can be used
(typically less than 100 daltons) at relatively
for final filtration or prefiltration, whereas a depth
high pressures using membranes with NMWLs of
filter is generally used in clarifying applications
1 kDa or lower. RO membranes are normally rated
where quantitative retention is not required or as
by their retention of sodium chloride while ultrafiltra-
a prefilter to prolong the life of a downstream
tion membranes are characterized according to
membrane. Membrane and depth filters offer
the molecular weight of retained solutes. Millipore
certain advantages and limitations. They can
water purification systems employ both reverse
complement each other when used together in a
osmosis membranes as well as ultrafiltration
microfiltration process system or fabricated device.
membranes. Reverse osmosis systems are primarily
The retention boundary defined by a membrane
used to purify tap water to purities that exceed
filter can also be used as an analytical tool to
distilled water quality. Ultrafiltration systems ensure
validate the integrity and efficiency of a system.
that ultrapure water is free from endotoxins as well
For example, in addition to clarifying or sterilizing
as nucleases for critical biological research.
filtration, fluids containing bacteria can be filtered to
trap the microorganisms on the membrane surface
for subsequent culture and analysis. Microfiltration
can also be used in sample preparation to remove
intact cells and some cell debris from the lysate.
Membrane pore size cut-offs used for this type of
separation are typically in the range of 0.05 m
to 1.0 m.

Figure 2. Ultrafiltration membranes vs. traditional microporous membranes
Ultrafiltration membranes
generally have two distinct
layers: a thin (0.11.5 m),
dense skin with a pore diameter
of 10 400 and a more
porous substructure. Any species
capable of passing through the
pores of the skin (whose size is
precisely controlled in manufac-
ture) can therefore freely pass
Cross-section of ultrafiltration
the membrane.
membrane with skin and porous
substructure.
O v e r v ie w o f M e m br a ne F ilt r at i o n M e m b r a n e P ro ce s s e s
Microporous membranes are
generally rigid, continuous
meshes of polymeric material
with defined pore sizes. They
are used to retain bacteria,
colloids and particulates.
Species are either retained
on the membrane surface or
trapped in its substructure.
Cross-section of traditional micro-
porous membrane with uniform
pore structure from top to bottom.

5
 Recovery
The ultimate aim of ultrafiltration is to maximize
recovery of solutes of interest, but there are many
membrane characteristics that affect that goal.
Factors affecting recovery include:
Nominal molecular weight limit (NMWL)/
nucleotide cut-off (NCO)
Retention
Concentration polarization
Flux
Nominal Molecular Weight Limit
A microfiltration membranes pore size rating,
typically given as a micron value, indicates that
particles larger than the rating will be retained.
Ultrafiltration membranes are rated according to
the nominal molecular weight limit (NMWL), also
sometimes referred to as molecular weight cut-off
(MWCO). The NMWL indicates that most dissolved
macromolecules with molecular weights higher than
the NMWL will be retained.
An ultrafiltration membrane with a stated NMWL
should retain (reject) at least 90% of a globular
solute of that molecular weight in daltons. However,
for a wider safety margin, the selected cut-off
O v e r v ie w o f M e m br a ne F ilt r at i o n Re cove r y
should be well below the molecular weight of the
solute to be retained. When solutes are to be
exchanged, the cut-off should be substantially
above that of the passing solute. A lower NMWL
increases rejection but decreases the filtration rate
for the same membrane material.
Retention and product recovery are a function
of a variety of other factors, including the molecular
shape and size of the molecule; electrical charac-
teristics; sample concentration and composition;
operating conditions; and device or system
configuration. Two membranes may have the
same NMWL but will exhibit different retention of
molecules within a relatively narrow range of sizes.
In addition, slender, linear molecules (e.g., nucleic
acids) may find their way through pores that will
retain a globular species of the same weight.
Retention can also be affected by hydration with
counter-ions. Nevertheless, NMWL has proven
to be an effective general indicator of membrane
performance for globular proteins.
6
When using membrane ultrafiltration for sample
concentration or desalting, care must be taken
to select a membrane (or device) with a NMWL
appropriate for the application. Because there
are several considerations in determining whether
a given solute will or will not be retained by a
membrane of a specific cut-off, it is best to choose
a device with cut-off at about one half of the
molecular weight of the protein to be concentrated.
This maximizes protein recovery and minimizes
filtration time.
Nucleotide Cut-off (NCO)
For most membranes, the NMWL is determined
experimentally under a standard set of operating
conditions. These analyses typically employ purified
globular proteins to serve as markers or indicators
of the retention characteristics of an ultrafiltration
membrane. Although this approach is useful for
choosing the appropriate NMWL for most protein
research applications, selection of a membrane
with an appropriate NMWL membrane for nucleic
acid or polysaccharide purification is considerably
more complex. By virtue of the rod-like three-
dimensional structures of these molecules, these
types of molecules require a tighter membrane
(with a smaller cut-off) than do globular proteins of
the same molecular weight. It is therefore convenient
to consider the membrane retention characteristics
of nucleic acids as being related to their length
(in nucleotides) rather than their molecular weight.
Complicating matters even further are several
additional factors that affect the recovery of nucleic
acid fragments from a membrane of a given
NMWL. These factors include: the strandedness
of the DNA or RNA molecule; whether the DNA is
linear, relaxed or supercoiled (for plasmid); the ionic
strength of the solvent; the velocity of the process
stream over the membrane; and the nature of the
driving force. The overall effect is that optimal
nucleic acid recovery is achieved in low salt buffers
run under conditions of relatively low velocity
(e.g., low vacuum pressure or low g-force).
The membrane and protocol developed for the
Montage PCR centrifugal filter device takes into
account these conditions in order to provide for high
recovery of small PCR products (e.g., ~150 base
pairs [bp]) as quickly as possible. For purifications
that are driven by vacuum, significantly tighter
membranes are typically required to obtain
optimal recovery.
If the DNA sample is in the presence of high salt
(or the device is run at a higher-than-recommended
g-force), a significantly reduced DNA recovery
may be observed. Under these conditions, higher
DNA or RNA recovery can be achieved by using a
tighter membrane. However, it will take significantly
longer to complete the purification. For applications
such as PCR where removal of unincorporated
single-stranded primers from double-stranded DNA
fragments is required, the molecular weights of
the primer and DNA fragment should differ by at
least an order of magnitude for efficient separation.
Millipore offers devices that are specifically
designed for separating and concentrating
genomic DNA and PCR products by ultrafiltration.
Retention
Retention, also sometimes called rejection, is a
function of molecular size and shape. Nominal
cut-off levels, defined with model solutes, are
convenient indicators. Degree of hydration, counter
ions, and steric effects can cause molecules with
similar molecular weights to exhibit very different
retention behavior. Many biological macromolecules
tend to aggregate, or change conformation under
varying conditions of pH and ionic strength, so that
effective size may be much larger than the native
molecule, causing increased rejection. Solute/
solvent and solute/solute interactions in the sample
can also change effective molecular size. For
example, some proteins will polymerize under
certain concentration and buffer conditions while
others (e.g., heme proteins) may break into
corresponding subunits. Ionic interactions or
stacking can cause small molecules to behave
similarly to molecules of greater molecular weight.
When this occurs, as in the case of phosphate ions
with a 500 NMWL membrane, the small molecules
may not effectively permeate the membrane.
Millipore recommends the selection of a mem-
brane filter NMWL that is one half the size of the
molecule of interest. Other manufacturers may
recommend a smaller differential between the size
of the NMWL and the size of the molecule but
Millipores recommendation is designed to provide
maximum recovery. Please see additional information
regarding membrane NMWL selection on page 14.
Concentration Polarization
Another factor affecting the retention characteristics
is the potential for membrane fouling, or concentra-
tion polarization. This occurs when there is an
accumulation of the retained solute on the surface
of the membrane. At high concentrations, a gel
layer forms that can act as a secondary membrane
(Figure 3). This may interfere with passage of the
molecules through the membrane and can adversely
affect the flow rate. In addition, pH, buffer compo-
nents, and concentration can result in a protein
behaving in an anomalous manner in terms of its
retention or passage by UF membranes.
During concentration polarization, the gel layer
on the membrane surface superimposes its own
rejection characteristics on those of the membrane.
Usually, concentration polarization increases
retention of lower-molecular weight species.
A membrane with a 100K NMWL may reject
1020% of albumin in a 0.1% solution of pure
albumin. However, in the presence of larger solutes
such as IgG, it may reject 90% of the albumin.
Concentration polarization makes it very difficult
to use UF for solute fractionation unless the solutes
O v e r v ie w o f M e m br a ne F ilt r at i o n Re cove r y
to be separated differ in size by at least an order
of magnitude.
Figure 3. High concentrations
Ultrafiltration separates proteins from soluble salts.
Concentration polarization slows down filtration.
The proteins form a gel layer on the membrane surface.
7
 Flux (UF Flow Rate)
During ultrafiltration, it is important to balance speed
with retention to obtain optimal performance. A
membranes flux is defined as the flow rate divided
by the membrane area. Using membranes with
higher NMWL ratings will increase the flow, but
at the same time lower the retention. A membrane
should be selected for required rejection, consistent
with desired flow rate. This is determined by surface
area, macrosolute type, solubility, concentration
and diffusivity, membrane type, temperature effects
on viscosity and, to some extent, pressure. When
concentration polarization is rate-controlling, flux
is affected by solute concentration, fluid velocity,
flow channel dimensions, and temperature.
Effects of Operating Parameters on Flux
Pressure
When ultrafiltering dilute protein solutions or colloid
suspensions, flux will increase with increasing
transmembrane pressure (TMP). These effects are
most apparent when operating under controlled
positive pressure, such as when using a stirred
cell. When the process is membrane-controlled
(i.e., when the resistance of the gel layer is much
smaller than that of the membrane), the flux-pressure
relationship is linear. When the process is controlled
by polarization (e.g., when the resistance of the
O v e r v ie w o f M e m br a ne F ilt r at i o n Re cove r y
gel layer is much larger than that of the membrane),
flux will reach a plateau and may actually decrease
with increases in pressure.
Concentration
When concentration of the retained species is very
low, flux is independent of concentration. As solute
concentration rises during operation, increased
viscosity and the polarization effect cause flux
to decrease.
Temperature
Increasing the operating temperature normally
increases UF rates. A higher temperature increases
solute diffusivity (typically 33.5% per degree
Celsius for proteins) and decreases solution
viscosity. Common practice is to operate at the
highest temperature tolerated by the solutes and
the equipment.
An exception to the rule is fermentation broth
concentration in the presence of some antifoams.
8
Many antifoams exhibit a phenomenon called
cloud point. As temperatures increase, antifoam
comes out of solution, forming a second phase.
Increasing temperature above the cloud point
causes flux to decrease.
pH
Changing solution pH often changes molecular
structure. This is especially true for proteins. At its
isoelectric point, a protein begins to precipitate,
causing a flux decrease.
Fouling
Flux decrease due to concentration polarization
should not be confused with the effect of membrane
fouling. Fouling is usually the deposition and
accumulation of submicron particles and solute on
the membrane surface and/or crystallization and
precipitation of smaller solutes on or within the
pores of the membrane. There may be a chemical
interaction with the membrane.
Importance of Recovery
While rejection is used to characterize membrane
performance, it does not always directly correlate
with solute recovery from a sample or volume.
Actual solute recoverythe amount of material
recovered after ultrafiltrationis generally based
on mass balance calculations.
In many cases, especially when working with
small samples of dilute, valuable solutions, the
degree of recovery of a target solute is vitally
important. In such cases, potential loss by
non-specific adsorption must be considered.
Different membrane materials adsorb biomol-
ecules to varying degrees. Where maximum
recovery is desired, the choice of a membrane
with the least non-specific adsorbtivity is essential.
Millipores Ultracel regenerated cellulose mem-
branes were specifically developed to minimize
non-specific adsorption.
Since adsorption is a direct function of membrane
and device surface area, device size must be
considered when recovery is important. Small, dilute
samples should be concentrated with membranes of
minimal surface area, commensurate with achieve-
ment of reasonable flow rates. Millipore offers a
wide range of centrifugal devices, stirred cells, and
tangential flow systems with an extensive choice of
membrane areas and NMWLs.
Mode of Operation
The pressure required for ultrafiltration can be Normal vs. Tangential Flow Filtration
supplied in a number of different ways depending
Filtration can be broken down into two different
on the product in use. For example, Millipores
operational modes: normal flow filtration (NFF)
small volume ultrafiltration products generally use
and tangential flow filtration (TFF). The difference
centrifugal force. Pump pressure is used with the
in fluid flow between these two modes is shown
tangential-flow filtration (TFF) products and com-
in Figure 4.
pressed gas is utilized with the stirred cell products.
In addition, Millipore provides multiwell ultrafiltration
products that utilize vacuum and centrifugation.

Figure 4. Normal flow filtration (NFF) vs. tangential flow filtration (TFF)
In normal flow filtration (NFF), fluid is convected directly toward the
membrane under an applied pressure. Particulates that are too large to
pass through the pores of the membrane accumulate at the membrane
surface or in the depth of the filtration media, while smaller molecules
pass through to the downstream side. This type of process is often
called dead-end filtration. However, the term normal indicates that
Normal Flow Filtration Tangential Flow Filtration
Feed Flow Pressure Pressure
the fluid flow occurs in the direction normal to the membrane surface,
so NFF is a more descriptive and preferred name. NFF can be used
for sterile filtration of clean streams, clarifying prefiltration, and virus/
protein separations.
In tangential flow filtration (TFF), the fluid is pumped tangentially
along the surface of the membrane. An applied pressure serves to
force a portion of the fluid through the membrane to the filtrate side.
As in NFF, particulates and macromolecules that are too large to
pass through the membrane pores are retained on the upstream side.
However, in this case the retained components do not build up at the
surface of the membrane. Instead, they are swept along by the tangen-
tial flow. This feature of TFF makes it an ideal process for finer sized-

O v e r v ie w o f M e m br a ne F ilt r at i o n M o d e of O p e r a t io n
based separations. Although TFF is more commonly associated with
Feed Flow
large scale processing, centrifugal UF devices with vertical membrane
panels, such as Amicon Ultra devices, also benefit from a TFF-like
mode of separation, particularly in a swinging bucket rotor. TFF is also
Membrane Membrane commonly called cross-flow filtration. However, the term tangential
is descriptive of the direction of fluid flow relative to the membrane,
so it is the preferred name.
Filtrate Filtrate

9
 Diafiltration
Millipore membranes provide an inexpensive means
of separating macromolecular mixtures into size-
graded classes either by direct ultrafiltration or by
diafiltration. Diafiltration removes microsolutes by
adding solvent to the solution being ultrafiltered
at a rate equal to the UF rate, independent of
microspecies concentration. This rapid, efficient
process washes microspecies from the solution at
constant volume, thereby purifying the retained
species. This process is most effective if the passing
molecules are at least 10 times smaller than the
molecules to be retained and concentrated by the
membrane. Diafiltration is useful for sample desalt-
ing and buffer exchange.
When diafiltration is used for sample desalting
or buffer exchange, there is no resulting change in
buffer composition. A solution volume with 100 mM
salt still contains 100 mM salt after the initial
concentration spin. Rediluting the retentate with water
and spinning again effectively decreases the salt
concentration of the sample by the concentration
factor of the ultrafiltration. For example, if a 4,000 L
sample containing 100 mM salt is concentrated to
50 L (80X) in an Amicon Ultra centrifugal filter unit,
O v e r v ie w o f M e m br a ne F ilt r at i o n D i af i l t r a t io n
rediluted with water to 4,000 L, and reconcen-
trated, the salt concentration will be reduced 80X
to 1.25 mM.
To achieve more complete salt removal, multiple
concentration and redilution spins are required. For
most samples, two concentration/reconstitution cycles
will remove about 99% of the initial salt content.
Figure 5. Removing salts from retained solutes using diafiltration
100 L
10 L
UF membrane
10X
concentration 90 L
of protein
100 mM 100 mM
NaCl NaCl
The sodium chloride concentration is reduced by dilution.
10
With very small sample volumes, dilution of the
sample before the initial concentration spin can
often decrease salt concentration to an acceptable
level. For example, if a 200 L sample containing
100 mM salt is diluted to 4,000 L before concen-
tration in an Amicon Ultra centrifugal filter unit, the
salt concentration in the 4,000 L sample will be
5 mM. The concentrate will still contain 5 mM
salt. If more complete salt removal is desired, a
re-dilution/spin cycle should be added. In this
example, if the original spin ended with 50 L of
retentate, redilution to 4,000 L results in 0.06 mM
salt concentration. The sample can then be recon-
centrated to 50 L in an Amicon Ultra centrifugal
filter device.
Diafiltration can be a continuous or a discontinu-
ous process. In continuous diafiltration, such
as in a stirred cell or a TFF device, the solution is
maintained at a fixed volume while solvent flows
continuously through the mixture. Salts and other
microsolutes are steadily removed by convective
transport. Microsolute exchange can be accom-
plished using the same principle. Constant operator
attention is not required and the possibility of solute
denaturation by overconcentration is eliminated.
In discontinuous diafiltration, such as in a centrifugal
ultrafiltration device, salts and microsolutes are
removed by repeated concentration and dilution
(Figure 5).
Add 90 L 100 L
10 L
Milli-Q water
10-fold 10X
dilution concentration 90 L
of protein
10 mM 10 mM
NaCl NaCl
Dialysis vs. Diafiltration
Dialysis is a traditional method for removing
microsolutes or exchanging solvents. It is a slow
diffusive process generally employing regenerated-
cellulose tubing as the barrier membrane. In dialysis,
the process solution and exchange solvent are
on opposite sides of the semi-permeable barrier
membrane through which permeating microsolutes
diffuse. The permeation rate of solutes from sample
to dialysate is a direct ratio to the solute concentra-
tion and inversely proportional to the solutes
molecular weight. Desalting by dialysis is time-
consuming and relatively inefficient at low
concentrations.
Millipores Amicon centrifugal concentrators
provide a fast, convenient, high-recovery alternative
to dialysis or precipitation without diluting samples.
The relative merits of diafiltration and dialysis are
summarized in the Table 1.
Fractionation
Fractionation is the process of separating a mixture
into its components using a combination of physico-
chemical properties of the solute. Ultrafiltration
membranes have been used for fractionation of
protein solutions on the basis of size1. This tech-
nique is also called membrane partitioning chroma-
tography1,2. Here, proteins larger than the pore size
are retained and those smaller than the pore size
pass through into the permeate. Since fractionation
is rarely absolute, acceptability of the results will
depend on whether the passing solute, retained
solute or both are of interest to the researcher and
their levels of purity and yield. Hence fractionation
efficiency is defined as a function of yield and purity
relative to the starting solution3.
Table 1. Comparison of diafiltration and dialysis
Diafiltration Dialysis
Transport convective with solvent, Transport diffusion-controlled,
independent of microsolute dependent on type of microsolute.
composition.
Rapid rate. Fractional removal Slow transport. Lower efficiency
independent of content. with decreased microsolute
concentration.
Ultrafiltration rate reduced Marked temperature dependence
with decreased temperature (reduced transport at lower
(net effect not as marked). temperature).
At elevated macrosolute content, Microsolute transport relatively
ultrafiltration rate reduced. unaffected by macrosolute content.
Minimal exchange solvent Frequent dialysate change.
required; easily contained in Recirculation about bags to
reservoir. maximize transport.
Simple automation with endpoint Automation possible with complex
control apparatus. equipment.
O v e r v ie w o f M e m br a ne F ilt r at i o n F r a c t io n a t io n
Factors to consider for efficient fractionation:
1. Selection of the appropriate molecular weight
cutoff: Selecting the appropriate MWCO of the
membrane is critical to ensure efficient fraction-
ation. The size of the retained and the passing
species affect the selection criteria for the
membrane pore size5.
1. Molecular weight of retained MWCO x 0.9
2. MWCO Molecular weight of passing x 3
3. Molecular weight of retained
Molecular weight of passing x 3
Example separation: Separating a 10 kDa
species from a 40 kDa protein is very likely to
succeed using a 30K MWCO membrane. This
is due to the fact that the retained solute is >0.9X
of the MWCO. Secondly the size difference
between the solutes is 4X and the ratio of the
MWCO to the passing solute is also 4X.
11
 In the case of separating an 8 kDa from a
24 kDa using a 30K MWCO, fractionation will
not be successful because the 24 kDa species is
only 80% of the pore size of the 30K MWCO
and it may leak into the permeate. Thus, careful
attention needs to be paid to the size of the
solutes and the available MWCOs.
If the application requires the retained species,
then the rules are reversed. For maximal purity,
the MWCO chosen should be very open, and for
maximal yield, the MWCO should be very tight.
2. Starting concentration of proteins: Starting
concentrations of protein solutions affect
fractionation efficiency4,5. When fractionating
with an Amicon Ultra device, the starting
concentration of the passing solute, to 10 mg/
mL does not affect yields or purity of the solute in
the permeate fraction. At the other end, for dilute
proteins, where the concentrations are below
0.5 mg/mL, the polarizing gel-like layer does
not foul the membrane. In this case we recom-
mend using the lowest MWCO possible to
prevent any trace amounts of retained solute
appearing in the filtrate.
Example separation: Consider the separation of
a 17 kDa protein from a 66 kDa protein. If a
MWCO of 50K is chosen (ratio of MWCO to
O v e r v ie w o f M e m br a ne F ilt r at i o n F r a c t io n a t io n
passing ~3X), yields and purity of the passing
solute are not affected even when the starting
concentration of the retained is greater than
5 mg/mL. However, if the 30K MWCO is
chosen to maximize purity of the passing solute,
then yields are significantly affected as concen-
tration of the retained increases to 5 mg/mL and
beyond. This is due to the fact that the ratio of
the MWCO to the passing solute is ~1.8X.
3. Multiple diafiltration steps to increase yields:
In order to increase yields of the passing solute
and to increase purity of the retained solute,
multiple spins are necessary. This approach is
also called as diafiltration, where the retentate
fraction is diluted back to the starting volume and
the sample is centrifuged again. For e.g., if 50%
of the passing solute was recovered in the filtrate
in the first spin, 50% of the remaining, i.e., 25%
would be recovered in a subsequent spin. The
total for 3 spins would be 50 + 25 + 12.5,
12
i.e. 87.5%. This relation holds true for the most
part since the total amount of the retained solute
is unchanged. With multiple spins, more of the
passing solute goes through into the permeate,
leaving the retained fraction purer and increasing
the yield of the passing solute.
4. A serial fractionation strategy for compartmen-
talizing unknown or complex mixtures:
Our recommendation for fractionation of
unknown mixtures is to start by separating the
proteins using the highest MWCO available.
The permeate fraction from this separation is
fractionated on the next highest MWCO and
so on, serially. Thus, the permeate fraction from
a device with a 100K MWCO membrane
contains proteins <100 kDa and has undetect-
able amounts of proteins with higher molecular
weights. Similarly, the permeate from the 50K
MWCO contains proteins <50 kDa and so on.
A point to note is that the efficiency of removing
proteins higher than the cutoff depends on the
size difference between the solute and the
MWCO, and the starting concentration of that
solute. Smaller solutes will pass through preferen-
tially compared to the larger ones.*
References
1. Blatt WF, Hudson BG, Robinson SM, Zipilivan
EM. Fractionation of protein solutions by
membrane partition chromatography.
Nature 1967;(216)511-513.
2. Blatt WF. Membrane partition chromatography:
a tool for fractionation of protein mixtures.
J Agric Food Chem 1971;19:589-594.
3. Ngiam SH, Bracewell DG, Zhou Y, Titchener-
Hooker NJ. Quantifying process tradeoffs in
the operation of chromatographic sequences.
Biotechnol Prog 2003;19:1315-1322.
4. Robertson BC, Zydney AL. Polarization and
adsorption effects on sieving in membrane
protein filtration. ASAIO Trans 1987;33:
118-122.
5. Nel RG, Oppenheim SF, Rodgers VG. Effects of
solution properties on solute and permeate flux
in bovine serum albumin-IgG ultrafiltration.
Biotechnol Prog 1994;10:539-542.
*Refer to A Simple Strategy for Protein Fractionation Using
Ultrafiltration for a protocol.
Membranes and Devices Membrane Selection . . . . . . . . . . . . . . . . . . . . 14

Device Selection . . . . . . . . . . . . . . . . . . . . . . . 16

for Ultrafiltration Microcon Centrifugal Filters . . . . . . . . . . . . . . . 17

Amicon Ultra-4 Centrifugal Filters . . . . . . . . . . 18

Amicon Ultra-15 Centrifugal Filters . . . . . . . . . . 20

Centriprep Centrifugal Filters . . . . . . . . . . . . . . 22

Centricon Plus-70 Centrifugal Filters . . . . . . . . . 23

MultiScreen Filter Plate . . . . . . . . . . . . . . . . . . 24

Ultrafree Centrifugal Filters . . . . . . . . . . . . . . . 25

Pellicon XL Cassettes and

Labscale TFF System . . . . . . . . . . . . . . . . . . . . 26

Pellicon 2 Mini Cassettes . . . . . . . . . . . . . . . . . 28

Prep/Scale Spiral Wound Filter Cartridges . . . . 29

Stirred Cells . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Ultrafiltration Discs . . . . . . . . . . . . . . . . . . . . . 31

e m br
Guid
M ct inD ge vMiceems br
e taoneSse laend a f ilt
s arnd
f oarneUlt ic ei os n M e m b r a n e P ro ce s s e s
D e rv at

13
 Membrane Selection
Millipore offers a complete range of centrifugal
devices used for sample concentration, purification,
and desalting or buffer exchange of soluble
macromolecules. Millipore products are also
available for applications such as cleaning up
PCR reactions, separating protein-bound from free
ligands, removing restriction enzymes, and recover-
ing oligonucleotides from agarose gels. This section
provides information to aid in choosing the correct
product for a particular application.
Millipore offers three distinct types of membranes
to choose from. This section will describe these
three types of membranes and then provide
information about choosing the correct membrane
based on typical recoveries or application.
Types of Membranes
Ultracel Ultrafiltration Membrane
M e m br a ne s a nd D e v ic e s f o r Ult r a f ilt r at i o n M e m b r a n e S e l e c t io n
To concentrate or desalt dilute solutions, use
Ultracel series regenerated cellulose ultrafiltration
membranes. The hydrophilic, tight microstructure
of Ultracel membranes assures the highest possible
retention with the lowest possible adsorption of
protein, DNA or other macromolecules.
Table 1. Membrane selection by recovery
Cytochrome c BSA IgG
NMWL (12.4 kDa) (67 kDa) (156 kDa)
3K Q Q Q the rule of two to ensure optimal recovery.
10K P Q Q
30K Q Q
50K P Q
100K P a NMWL at least two times smaller than the
14
Biomax Ultrafiltration Membrane
To concentrate or desalt higher volumes of more
concentrated samples (recommended for protein
concentrations greater than 1.0 mg/mL), use
Biomax polyethersulfone (PES) ultrafiltration mem-
branes. Biomax membranes are recommended for
samples such as serum, plasma, or conditioned
tissue culture media.
Durapore Microporous Membrane
To clarify biological samples, recover DNA from
agarose gels, retain chromatography resins or
suspended solid media, use Durapore hydrophilic
PVDF microporous membranes. Durapore mem-
branes allow all soluble protein and nucleic acids
to pass, retaining sub-cellular fragments, whole cell
and particulate materials. Durapore membranes are
extremely hydrophilic, and they provide the lowest
binding of proteins and other biologicals of all
commercially available microporous membranes.
Membranes Organized by Typical Recoveries
For globular proteins, there is a good correlation
between molecular weight and Stokes radius.
This usually allows one to predict the recovery of a
protein based on its molecular weight if a membrane
with the same nominal molecular weight rating is
used (see typical recovery of a panel of protein
solutes in Table 1). However, in order to accommo-
date the wide range of potential protein solutes with
different tertiary structures, we suggest initially using
Rule of Two
For Ultracel (regenerated cellulosic) membranes,
Millipore recommends using a membrane with
molecular weight of the protein solute that one
intends to concentrate.
Rule of Three
For Biomax (polyethersulfone) membranes for
stirred cells and TFF, Millipore recommends using
a membrane with a NMWL at least three times
smaller than the molecular weight of the protein
solute that one intends to concentrate.
Membranes Organized by Application
See Table 2 to determine which centrifugal product
to use based on application and membrane.

Microporous
Table 2. Membrane selection by application Ultrafiltration Membranes Membranes Specialty
Molecular Weight Devices
Type* Pore Size (m)
(NMWL)

Montage Gel Extraction

Micropure-EZ
Montage PCR
Ultracel
Biomax

100K

0.45
0.65
30K
50K
10K

0.2

5.0
0.1
3K
5K
Protein concentration
Protein purification/desalting/buffer exchange

M e m br a ne s a nd D e v ic e s f o r Ult r a f ilt r at i o n M e m b r a n e S e l e c t io n
Desalting of column fractions
Protein isolation from cell lysates
Peptide concentration/desalting/buffer exchange
Antibody concentration
Virus concentration or removal
Nucleic acid concentration/desalting/buffer exchange
Oligonucleotide concentration/desalting/buffer exchange
PCR cleanup
Remove linkers prior to cloning
Remove labeled nucleotides
Antibody purification from hybridoma cells
Rapid restriction mapping
Clarify samples of particulate prior to HPLC
Clarification of cell lysates and tissue homogenates
Cell harvesting
Natural product screening
Restriction enzyme removal
Bound vs. free drugs from serum/plasma (protein removal)
DNA/RNA recovery from polyacrylamide gel
DNA recovery from agarose gel
Oligonucleotide recovery from polyacrylamide gel
Removal of unincorporated label (e.g., fluorescein) from protein
Removal of imidazole from His-tag fusion protein
*Selection of ultrafiltration membrane: Ultracel regenerated cellulose membrane is ideal for protein samples and nucleic acids. Biomax polysulfone
membrane is ideal for complex samples (e.g., serum).

15
 Device Selection
See Table 3 to determine which centrifugal product
to use for protein concentration based
on initial sample molecular weight and volume.

Table 3. Protein concentration devices by filtration capacity

Filtration Capacity
Membrane
0.5 2 4 15 20 70 1 2 10 10
Type
Millipore Device mL mL mL mL mL mL L L L L
Small Volume Filtration Devices
Microcon Centrifugal Filters* U
Ultrafree-MC Centrifugal Filters M
MultiScreen Filter Plate with Ultracel Membrane** U
Medium Volume Filtration Devices
Ultrafree-CL Centrifugal Filters M
Amicon Ultra-4 Centrifugal Filters U
Amicon Ultra-15 Centrifugal Filters U
Centriprep Centrifugal Filters U
Large Volume Filtration Devices
M e m br a ne s a nd D e v ic e s f o r Ult r a f ilt r at i o n D ev ice S e l e c t io n

Centricon Plus-70 Centrifugal Filters U

Amicon Stirred Cells
Series 8000, High Output, Solvent-Resistant Stirred Cells U
Tangential Flow Filtration (TFF)
Pellicon XL 50 Ultrafiltration Devices U
Prep/Scale Spiral Wound UF Modules

U
Pellicon 2 Ultrafiltration Modules U
U = Ultrafiltration M = Microporous
*Bold type indicates recommended devices.
**96-well plate. Volume per well.

16
Microcon Centrifugal Filters

mL
0.5
Microcon centrifugal filters are the lab standard
for small volume concentration.
They allow you to process macromolecular Highest
solutions up to 500 L using any centrifuge that can Recovery
accept 1.5 mL tubes. The devices low-adsorption
Ultracel YM membrane and patented invert
recovery spin combine to yield unusually high
recovery ratestypically >95% of the sample,
with concentration factors as high as 100X.
Maximum starting volume: 500 L
Typical sample concentration volume: 515 L
Low-binding Ultracel-YM regenerated
cellulose membrane
Solute recoveries typically >95%

M e m br a ne s a nd D e v ic e s f o r Ult r a f ilt r at i o n M icro co n C e n t rif u g a l F i l te r s

Typical Protein Recovery
Retentate Recovery (%) by Nominal MW
Solute (Concentration) MW 3,000 10,000 30,000 50,000 100,000
Cytochrome c (0.25 mg/mL) 12,400 94 95
-Chymotrypsinogen (1 mg/mL) 25,000 95
Ovalbumin (1 mg/mL) 45,000 95
Bovine Serum Albumin (1 mg/mL) 67,000 95 95
Bovine IgG Fraction II (1 mg/mL) 156,000 95 95

Protocol
Ordering Information
Description NMWL Qty/Pk* Catalogue No.
Microcon Filter Units 3,000 24 42403
Add
Sample 100 42404
10,000 24 42406
100 42407
30,000 24 42409
100 42410
Invert into 50,000 24 42415
Receiver 100 42416
100,000 24 42412
100 42413

*Additional package sizes available. Contact Millipore.

Recover
Sample

For additional information, visit www.millipore.com/microcon

17
 Amicon Ultra-4 Centrifugal Filters
1 4 m L

Amicon Ultra-4 Centrifugal Filters set the perfor-

mance standard for concentrating small-volume
samples. Ultracel regenerated cellulose low-binding
ultrafiltration membrane combines with a vertical
housing for fast sample processing with high
recovery. Ultra
Processes 24 mL in as few as 10 minutes Recovery,
Ultra
>90% typical sample recoveries Speed

Compatible with most rotor types

Double membrane panels increase surface
area and reduce filtration times
Deadstop prevents sample from spinning
M e m br a ne s a nd D e v ic e s f o r Ult r a f ilt r at i o n A m ico n U l t r a - 4 C e n t rif u g a l F i l te r s

to dryness and eliminates the need for an

inverse spin
-marked for in vitro diagnostic use
(10K NMWL)

Typical Spin Times Comparative Performance

Spin conditions: 4000 x g, swinging bucket rotor at 100
25 C, 4 mL sample. 3K and 10K: Cytochrome c,
0.25 mg/mL; 30K and 50K: BSA, 1 mg/mL; 80
Recovery (%)

100K: lgG, 1 mg/mL.

60
4.0
40
3.5
Filtrate Volume (mL)

3.0 20
2.5
0
2.0 3,000 NMWL Brand V Brand PG Brand P Amicon Ultra-4
1.5 10,000 NMWL
30,000 NMWL Devices with 10 kDa* NMWL using Cytochrome c
1.0 50,000 NMWL
0.5 100,000 NMWL
(0.025 mg/mL).
0
0 5 10 15 20 25 30
Spin Time (min)

Complex sample volumes of 4 mL can be concen-

trated or diafiltered in as few as 15 minutes.

*Brand PG has a 9K membrane.

18
Typical Protein Recovery

1 4 m L
Retentate Recovery (%) by Nominal MW
Solute (Concentration) MW 3,000 10,000 30,000 50,000 100,000
Cytochrome c (0.25 mg/mL) 12,400 94 95
Bovine Serum Albumin (1 mg/mL) 67,000 95 94
IgG (1 mg/mL) 156,000 91
Typical recoveries for 4 mL starting volume in fixed angle rotor at 7500 x g at 25 C. Spin times: 5K (20 minutes); 10 and 30K (10 minutes);
50K (5 minutes); 100K (15 minutes).

Protocol
Ordering Information
Description NMWL Qty/Pk* Catalogue No.
Amicon Ultra-4 Centrifugal Filters 3,000 8 UFC8 003 08
are assembled with 24 UFC8 003 24
centrifuge tubes and caps 96 UFC8 003 96
Add
10,000 8 UFC8 010 08
Sample
24 UFC8 010 24

M e m br a ne s a nd D e v ic e s f o r Ult r a f ilt r at i o n A m ico n U l t r a - 4 C e n t rif u g a l F i l te r s

96 UFC8 010 96
30,000 8 UFC8 030 08
24 UFC8 030 24
96 UFC8 030 96
50,000 8 UFC8 050 08
24 UFC8 050 24
96 UFC8 050 96
100,000 8 UFC8 100 08
24 UFC8 100 24
96 UFC8 100 96

*Additional package sizes available. Contact Millipore.

Recover
Purified
Sample

For additional information, visit www.millipore.com/ultra4

19
 Amicon Ultra-15 Centrifugal Filters
mL
15

Amicon Ultra-15 Centrifugal Filters are the premier

devices for concentrating mid-volume samples.
They incorporate low-binding Ultracel regenerated
Ultra
cellulose ultrafiltration membrane and a vertical Recovery,
design for maximum recovery and minimal spin times. Ultra
Speed
Processes up to 15 mL in as few as 10 minutes
>90% typical sample recoveries
Compatible with most rotor types
Double membrane panels increase surface area
and reduce filtration times
Deadstop prevents sample from spinning to dry-
ness and eliminates the need for an inverse spin
M e m br a ne s a nd D e v ic e s f o r Ult r a f ilt r at i o n A m ico n U l t r a -15 C e n t rif u g a l F i l te r s

-marked for in vitro diagnostic use

(10K NMWL)

Typical Spin Times Comparative Performance

Spin conditions: 4000 x g, swinging bucket rotor at 100
25 C, 15 mL sample. 3K and 10K: Cytochrome c,
0.25 mg/mL; 30K and 50K: BSA, 1 mg/mL; 80
Recover (%)

100K: lgG, 1 mg/mL.

60
16
40
14
Filtrate Volume (mL)

12 20
10
0
8 3,000 NMWL Brand V Brand PG Brand P Amicon Ultra-15
6 10,000 NMWL
30,000 NMWL Devices with 10 kDa* NMWL using Cytochrome c
4 50,000 NMWL
2 100,000 NMWL
(0.025 mg/mL).
0
0 5 10 15 20 25 30 35 40 45 50 55 60 65
Spin Time (min)

Complex sample volumes of 15 mL can be concen-

trated or diafiltered in as few as 15 minutes.

*Brand PG has a 9K membrane.

20
Typical Protein Recovery

mL
Retentate Recovery (%) by Nominal MW

15
Solute (Concentration) MW 3,000 10,000 30,000 50,000 100,000
Cytochrome c (0.25 mg/mL) 12,400 91 93
Bovine Serum Albumin (1 mg/mL) 67,000 98 93
IgG (1 mg/mL) 156,000 89
Typical recoveries for 15 mL starting volume in swinging bucket rotor at 400 x g at 25 C. Spin times: 5K (45 minutes);
10 and 100K (20 minutes); 30K (10 minutes); 50K (15 minutes).

Protocol
Ordering Information
Description NMWL Qty/Pk* Catalogue No.
Amicon Ultra-15 Centrifugal 3,000 8 UFC9 003 08
Filters are assembled with 24 UFC9 003 24
Add centrifuge tubes and caps 96 UFC9 003 96
Sample
10,000 8 UFC9 010 08
24 UFC9 010 24

M e m br a ne s a nd D e v ic e s f o r Ult r a f ilt r at i o n A m ico n U l t r a -15 C e n t rif u g a l F i l te r s

96 UFC9 010 96
30,000 8 UFC9 030 08
24 UFC9 030 24
96 UFC9 030 96
50,000 8 UFC9 050 08
24 UFC9 050 24
96 UFC9 050 96
100,000 8 UFC9 100 08
24 UFC9 100 24
96 UFC9 100 96

Recover *Additional package sizes available. Contact Millipore.

Purified
Sample

For additional information, visit www.millipore.com/ultra15

21
 Centriprep Centrifugal Filters
5 15 m L

Use Centriprep Centrifugal Filters to concentrate

and desalt high solute biological samples in the
For
515 mL volume range. The devices are compatible High Solute
with most centrifuges that accommodate 50 mL Samples
centrifuge tubes.
Unique inverse flow mode of operation
with large deadstop
Starting volume from 515 mL
Low-binding Ultracel regenerated
cellulose membrane
Fast sample processing with typical
recovery of >90%

Typical Protein Recovery

M e m br a ne s a nd D e v ic e s f o r Ult r a f ilt r at i o n C e n t ri p re p C e n t rif u g a l F i l te r s

Retentate Recovery (%)

Solute (Concentration) Nominal MW 3,000 10,000 30,000 50,000
Cytochrome c (0.25 mg/mL) 12,400 90 85
-Chymotrypsinogen (1 mg/mL) 25,000 90
Ovalbumin (1 mg/mL) 45,000 90
Bovine Serum Albumin (1 mg/mL) 67,000 90
Bovine IgG Fraction II (1 mg/mL) 156,000 90

Protocol
Ordering Information
Description Membrane NMWL Qty/Pk* Catalogue No.

Disassemble Centriprep 3,000 3,000 24 4302

Device and Centrifugal Filters 96 4303
Add Sample 10,000 10,000 24 4304
96 4305
Reassemble 30,000 30,000 24 4306
and Place in 96 4307
Centriprep
50,000 50,000 24 4310
96 4311
Centrifuge Until
Equilibrium is *Additional package sizes available. Contact Millipore.
Reached

Decant Filtrate
and Centrifuge
Again

Disassemble and
Recover Retentate

22
Centricon Plus-70 Centrifugal Filters

30 70 m L
The Centricon Plus-70 device can concentrate
most 70 mL solutions down to 350 L in less than 25
Convenient
minutes, making it a convenient alternative to stirred Alternative
cells. Typical sample recoveries are >90% with minimal to Stirred
sample loss due to non-specific binding. Cells

High concentration factors, with samples typically

concentrated in the 50X to 200X range
Low hold-up volume
Invert spin method of recovery
Low binding Ultracel regenerated cellulose mem-
brane
Low-binding polypropylene housing

M e m br a ne s a nd D e v ic e s f o r Ult r a f ilt r at i o n C e n t rico n P lu s -70 C e n t rif u g a l F i l te r s

Typical Protein Recovery
Retentate Recovery (%)
Solute (Concentration) Nominal MW 3,000 10,000 30,000 50,000
Cytochrome c (0.25 mg/mL) 12,400 94
Bovine Serum Albumin (1 mg/mL) 67,000 85 95 96 84
IgG Fraction II (1 mg/mL) 156,000 94 91 91

Protocol Typical Spin Times

80
Filtrate Volume (mL)

60
Add Sample

40 PBS
0.25 mg/mL BSA
1.0 mg/mL BSA
20 5.0 mg/mL BSA

0
0 10 20 30 40
Time (min)
Invert into
Receiver 5K NMWL Biomax membrane spun at 3500 x g in a
swinging bucket rotor.

Ordering Information
Recover Purified
Sample Description Membrane NMWL Qty/Pk* Catalogue No.
Centricon Plus-70 Biomax 5,000 8 UFC7 005 08
Centrifugal Filters Ultracel 10,000 8 UFC7 010 08
30,000 8 UFC7 030 08
100,000 8 UFC7 100 08

*Additional package sizes available. Contact Millipore.

For additional information, visit www.millipore.com/centricon70

23
 MultiScreen Filter Plate with
mL
< 0.5

Ultracel-10 Membrane
The first automation high throughput ultrafiltration
plate for protein purification. 10,000 NMWL
Ultracel regenerated cellulose membrane provides
low non-specific binding and high protein recovery.
Processes from 50 to 500 L
95% typical retention of Cytochrome c
M e m br a ne s a nd D e v ic e s f o r Ult r a f ilt r at i o n M u l t i S cre e n F i l te r P l a te w i t h U l t r a ce l -10 M e m b ra n e

Compatible with standard microtiter receiver

plates (300 L, 700 L deep well, 150 L
conical bottom)
Fast, easy centrifugal protocol
Ideally suited for instrumentation and
liquid handling equipment
Uniform performance from well to well

Typical Spin Times

300
Typical Protein Recovery 250
Buffer
Cytochrome c (1 mg/mL
Filtrate Volume (L)

BSA (1 mg/mL)
Membrane Protein Typical Protein 200 Fetal Bovine Serum
NMWL Solute Retention (%) 150
10,000 Cytochrome c, 95
100
12,400 daltons
(1 mg/mL) 50

10,000 BSA, 99 0
0 10 20 30 40 50 60
67,000 daltons Spin Time (min)
(1 mg/mL)
300 L sample spun at 2000 x g in swinging
300 L sample spun at 2000 x g in swinging bucket rotor.
bucket rotor.

Ordering Information
Description Qty/Pk* Catalogue No.
MultiScreen Filter 10 MAUF 010 10
Plate with Ultracel-10
membrane

*Additional package sizes available. Contact Millipore.

For additional information, visit www.millipore.com/ultracel

24
Ultrafree Centrifugal Filters

0.12 m L
for Sample Clarification
Ultrafree-MC Centrifugal Filters
For sample clarification with low hold-up
Maximum starting volume: 500 L
Hold-up volume: <5 L

Ultrafree-CL Centrifugal Filters

For sample clarification with high recovery
Maximum starting volume: 2 mL
Hold-up volume: <10 L Ultrafree-MC

Ordering Information

M e m br a ne s a nd D e v ic e s f o r Ult r a f ilt r at i o n Ul t r af re e C e n t rif u g a l F i l te r s

Description Membrane Pore Size (m) Sterility Qty/Pk Catalogue No.
Ultrafree-MC Durapore (PVDF) 0.1 Non-sterile 25 UFC3 0VV 25
Centrifugal 100 UFC3 0VV 00
Filters 0.22 Non-sterile 25 UFC3 0GV 25
100 UFC3 0GV 0S
Sterile 50 UFC3 0GV 05
0.45 Non-sterile 25 UFC3 0HV 25
Non-sterile 100 UFC3 0HV 00
Sterile 50 UFC3 0HV 0S
0.65 Non-sterile 25 UFC3 0DV 25
Ultrafree-CL
100 UFC3 0DV 00
Sterile 50 UFC3 0DV 0S
5.0 Non-sterile 100 UFC3 0SV 00
Protocol Hydrophilic PTFE 0.22 Non-sterile 25 UFC3 0LG 25
0.45 Non-sterile 25 UFC3 0LH 25
Ultrafree-CL Durapore (PVDF) 0.1 Non-sterile 25 UFC4 0VV 25
Add Centrifugal 100 UFC4 0VV 00
Sample Filters 0.22 Non-sterile 25 UFC4 0GV 25
100 UFC4 0GV 00
0.45 Non-sterile 25 UFC4 0HV 25
100 UFC4 0HV 00
Close 0.65 Non-sterile 25 UFC4 0DV 25
Lid
5.0 Non-sterile 25 UFC4 0SV 25
Hydrophilic PTFE 0.22 Non-sterile 25 UFC4 0LG 25
0.45 Non-sterile 25 UFC4 0LH 25

Additional package sizes available. Contact Millipore.

Collect
Particulates
Sample

Filtrate

25
 Pellicon XL Cassettes and
12 L

Labscale TFF System

Pellicon XL cassettes allow you to easily concentrate
or diafilter samples using tangential flow filtration.
The cassettes are available with either microfiltration
or ultrafiltration membranes.
Use cassettes with Ultracel regenerated
cellulose or Biomax polyethersulfone
(ultrafiltration) membranes for
concentrating, desalting proteins,
M e m br a ne s a nd D e v ic e s f o r Ult r a f ilt r at i o n Pe l l ico n X L C a s s e t te s a n d L a b s c a l e T F F Sy s te m

polysaccharides, lipid solutions,

cell suspensions, and mammalian cells.
Use cassettes with Durapore PVDF
(microporous) membrane for sample preparation,
washing, or cell harvesting, or for clarifying cell
cultures, lysates, or fermentation broths.
The Labscale TFF system is specifically designed
to operate Pellicon XL modules. The system reservoir
accepts direct docking of the device, eliminating the
need for tubing connections.
Provides the lowest working volumes, with typical
sample concentrations of >25X
An optional 100 mL reservoir is available for
low volume applications

Typical Processing Times

Pellicon XL
150

125
Process Flux (Lmh)

100

75

50

25

0
0 1 10 100
Protein Concentration (g/L)

Pellicon XL cassettes provide rapid, efficient concen-

tration of protein-containing solutions. Data show
concentration of BSA with a 10K NMWL cassette
with Ultracel regenerated cellulose membrane on a
LabScale TFF system operating at 13 C with 40 psi
inlet pressure and 20 psi outlet pressure.

26
Ordering Information

12 L
Description Membrane NMWL Catalogue No.
Pellicon XL Ultracel Regenerated 5,000 PXC0 05C 50
Cassettes Cellulose 10,000 PXC0 10C 50
30,000 PXC0 30C 50
300,000 PXC3 00C 50
1,000,000 PXC0 1MC 50
Biomax 5,000 PXB0 05A 50
Polyethersulfone 8,000 PXB0 08A 50
10,000 PXB0 10A 50
30,000 PXB0 30A 50
50,000 PXB0 50A 50
100,000 PXB1 00C 50
300,000 PXB3 00C 50 Multi-Manifold accessory allows

M e m br a ne s a nd D e v ic e s f o r Ult r a f ilt r at i o n Pe l l ico n X L C a s s e t te s a n d L a b s c a l e T F F Sy s te m

500,000 PXB5 00C 50 up to three cassettes to be mounted
on the Labscale system for processing
1,000,000 PXB0 1MC 50
multi-liter samples.

Description Membrane Pore Size (m) Catalogue No.

Pellicon XL Durapore PVDF 0.10 PXVV PPC 50
Cassettes 0.22 PXGV PPC 50
0.45 PXHV MPC 50
0.65 PXDV PPC 50

Description Pore Size (m) Catalogue No.

System kit includes pump module, stir base, 500 mL and
100 mL reservoirs, stand for 100 mL reservoir, and multi-manifold.
Labscale TFF System 115 V XX42 LSS 11
230 V XX42 LSS 12
GB-230 V XX42 LSS 13

27
 Pellicon 2 Mini-Cassettes
L
10

Cassette-style Pellicon 2 modules are available

with Durapore PVDF (microporous) and Ultracel
regenerated cellulose and Biomax polyethersulfone
(ultrafiltration) membranes for high performance
in large volume concentration and diafiltration
applications, as well as scale-up applications.
Cassettes for low viscosity (A-screen), low to
medium viscosity (C-screen), and high viscosity/
high concentration (V-screen) solutions.
Larger Pellicon cassette systems are available
for processing 250 L or more. Contact Millipore
or visit our web site for more information.
Stainless steel Pellicon 2 Mini-Cassette Holder
can operate up to three mini-cassettes.

Ordering Information
M e m br a ne s a nd D e v ic e s f o r Ult r a f ilt r at i o n Pe l l ico n 2 M i n i - C a s s e t te s

Description Membrane NMWL A Screen C Screen V Screen

Pellicon 2 Biomax 5,000 P2B0 05A 01 P2B0 05V 01
Mini-Cassettes Polyethersulfone 8,000 P2B0 08A 01 P2B0 08V 01
10,000 P2B0 10A 01 P2B0 10V 01
30,000 P2B0 30A 01 P2B0 30V 01
50,000 P2B0 50A 01 P2B0 50C 01 P2B0 50V 01
100,000 P2B1 00A 01 P2B1 00C 01 P2B1 00V 01
300,000 P2B3 00C 01 P2B3 00V 01
500,000 P2B5 00C 01 P2B5 00V 01
1,000,000 P2B0 1MC 01 P2B0 1MV 01
Ultracel 5,000 P2C0 05C 01 P2C0 05V 01
Regenerated 10,000 P2C0 10C 01 P2C0 10V 01
Cellulose 30,000 P2C0 30C 01 P2C0 30V 01
100,000 P2C1 00C 01 P2C1 00V 01
300,000 P2C3 00C 01 P2C3 00V 01
1,000,000 P2C0 1MC 01 P2C0 1MV 01

Accessories
Description Catalogue No.
Pellicon 2 Mini Holder XX42 PMI NI
Pellicon 2 Mini Holder Fitting Kit XX42 PFK 01

28
Prep/Scale Spiral Wound
Filter Cartridges
Prep/Scale spiral wound filter cartridges are
available in three sizes for easy, reliable preparation
of samples ranging from 100 liters down to 100 mL.
Self-contained design ensures leak-free filtration
Hose barb connectors for easy setup
Can be operated with standard 1 to
16 L/minutes peristaltic pumps
Prep/Scale Holder includes fittings and basic

M e m br a ne s a nd D e v ic e s f o r Ult r a f ilt r at i o n P re p / S c a l e S p i r a l Wo u n d F i l te r C a r t ri d g e s
instrumentation

Three Cartridge Sizes

Prep/Scale- Prep/Scale- Prep/Scale-
TFF-1 TFF-2 TFF-6
Filter Area (m2) 0.1 0.23 0.54
Minimum Working 100 150 250
Volumes (mL)*
*Including pump tubing

Ordering Information
Description Membrane NMWL Prep/Scale-TFF-1 Prep/Scale-TFF-2 Prep/Scale-TFF-6
Prep/Scale Spiral Regenerated 1,000 CDUF 001 LA CDUF 002 LA CDUF 006 LA
Wound Filer Cellulose 3,000 CDUF 001 LB CDUF 002 LB CDUF 006 LB
Cartridges 5,000 CDUF 001 LC CDUF 002 LC CDUF 006 LC
10,000 CDUF 001 LG CDUF 002 LG CDUF 006 LG
30,000 CDUF 001 LT CDUF 002 LT CDUF 006 LT
100,000 CDUF 001 LH CDUF 002 LH CDUF 006 LH
300,000 CDUF 001 LM CDUF 002 LM CDUF 006 LM
Polyethersulfore 10,000 CDUF 001 TG CDUF 002 TG CDUF 006 TG
30,000 CDUF 001 TT CDUF 002 TT CDUF 006 TT
50,000 CDUF 001 TQ CDUF 002 TQ CDUF 006 TQ
100,000 CDUF 001 TH CDUF 002 TH CDUF 006 TH
300,000 CDUF 001 TM CDUF 002 TM CDUF 006 TM

Accessories
Description Catalogue No.
Prep/Scale Module Holder XX42 PS0 01
Peristaltic Pumps for TFF-1 use XX8200
for TFF-2 and TFF-6 use XX80 EL

29
 Stirred Cells
Concentrate, diafilter, and exchange buffers
for macromolecule solutions including proteins,
enzymes, antibodies and viruses.

Series 8000 Stirred Cells

Five different sizes handle volumes from
3 mL to 400 mL
High flow rates with solutions up to
10% macrosolute concentration

Solvent-Resistant Stirred Cells

Two sizes: 75 mL (47 mm disc) and
300 mL (76 mm disc)
Borosilicate glass cylinder and PTFE Series 8000 Stirred Cell

components for broad chemical compatibility

Series 8000 Models

l s ce s s e s

Model 8003 Model 8010 Model 8050 Model 8200 Model 8400
b r adn eC ePl ro

Maximum Process Volume (mL) 3 10 50 200 400

Minimum Process Volume (mL) 0.075 1.0 2.5 5.0 10.0
e mi rre

Membrane Diameter (mm) 25 25 44.5 63.5 76

ic ei os n M St

Effective Membrane Area (cm ) 2

0.9 4.1 13.4 28.7 41.8
Hold-up Volume (mL) 0.07 0.2 0.5 1.2 1.5
D e rv at
a f ilt

Ordering Information
s arnd

Description Catalogue No.

f oarneUlt

Series 8000 Stirred Cells Model 8003 5125

Model 8010 5121
ct inD ge vMiceems br

Model 8050 5122

Model 8200 5123
Model 8400 5124
Solvent-Resistant Stirred Cells For 47 mm membranes XFUF 047 01
e taoneSse laend

For 76 mm membranes XFUF 076 01

Solvent-Resistant
Stirred Cell
e m br
Guid
M

30
Ultrafiltration Discs
High Recovery Ultracel Ultrafiltration Membranes
Hydrophilic, tight microstructure for highest
possible retention with lowest possible protein
adsorption
For use when concentrating or desalting
extremely dilute solutions or whenever your
sample is hydrophobic

High Flow Biomax Ultrafiltration Membranes

Open microstructure speeds concentration
To concentrate and desalt higher volumes or
more concentrated samples

Ordering Information

M e m br a ne s a nd D e v ic e s f o r Ult r a f ilt r at i o n Ul t r af i l t r a t io n D i s c s
Ultracel Ultrafiltration Discs
Regenerated Cellulose
NMWL
Filter
Diameter (mm) Qty/Pk 1,000 3,000 5,000 10,000 30,000 100,000
25 10 PLAC 025 10 PLBC 025 10 PLCC 025 10 PLGC 025 10 PLTK 025 10 PLHK 025 10
44.5 10 PLAC 043 10 PLBC 043 10 PLCC 043 10 PLGC 043 10 PLTK 043 10 PLHK 043 10
47 10 PLAC 047 10 PLBC 047 10 PLCC 047 10 PLGC 047 10 PLTK 047 10 PLHK 047 10
63.5 10 PLAC 062 10 PLBC 062 10 PLCC 062 10 PLGC 062 10 PLTK 062 10 PLHK 062 10
76 10 PLAC 076 10 PLBC 076 10 PLCC 062 10 PLGC 076 10 PLTK 076 10 PLHK 076 10
90 5 PLAC 090 05 PLBC 090 05 PLCC 090 05 PLGC 090 05 PLHK 090 05 PLHK 090 05
150 5 PLAC 150 05 PLBC 150 05 PLCC 150 05 PLGC 150 05 PLTK 150 05 PLHK 150 05

Biomax Ultrafiltration Discs

Polyethersulfone
NMWL
Filter
Diameter (mm) Qty/Pk 5,000 10,000 30,000 50,000 100,000
25 10 PBCC 025 10 PBGC 025 10 PBTK 025 10 PBQK 025 10 PBHK 025 10
44.5 10 PBCC 043 10 PBGC 043 10 PBTK 043 10 PBQK 043 10 PBHK 043 10
47 10 PBCC 047 10 PBGC 047 10 PBTK 047 10 PBQK 047 10 PBHK 047 10
63.5 10 PBCC 062 10 PBGC 062 10 PBTK 062 10 PBQK 062 10 PBHK 062 10
76 10 PBCC 076 10 PBGC 076 10 PBTK 076 10 PBQK 076 10 PBHK 076 10
90 5 PBCC 090 05 PBGC 090 05 PBTK 090 05 PBQK 090 05 PBHK 090 05
150 5 PBCC 150 05 PBGC 150 05 PBTK 150 05 PBQK 150 05 PBHK 150 05

31
32
Guid e t o S e l e ct in g M e m br a ne s a nd D e v ic e s
Protocols
Concentration, Desalting and Buffer Exchange . 34

Detergent Removal . . . . . . . . . . . . . . . . . . . . 38

for Proteins Two-Dimensional Electrophoresis

Sample Preparation . . . . . . . . . . . . . . . . . . . . 40

Purification of Serum Peptides for Biomarker . . . 42

Rapid Antibody Concentration . . . . . . . . . . . . . 45

Affinity Purification . . . . . . . . . . . . . . . . . . . . . 46

Removal of Unincorporated Label

from Labeled Protein . . . . . . . . . . . . . . . . . . . . 50

Rapid Purification of Monoclonal Antibodies . . . 52

A Simple Strategy for Protein Fractionation . . . . 54

Urine Concentration . . . . . . . . . . . . . . . . . . . . 56
Use of Centrifugal Filter Devices as an
Alternative to Stirred Cells . . . . . . . . . . . . . . . . 58

P ro t o c o l s f o r P ro t e in s

33
 Concentration, Desalting, and
Buffer Exchange with Amicon Ultra
or Microcon Centrifugal Filters
Introduction
Amicon centrifugal devices from Millipore are ideal
for removal or exchange of salts, sugars, nucleo-
tides, and non-aqueous solvents, as well as other
materials of low molecular weight. They also serve
to separate free from bound species.
Millipore centrifugal concentrators provide fast,
convenient, high-recovery alternatives to dialysis
and ethanol precipitation. Sample dilution, often
associated with spin columns, is not a problem.
Salt transfer across the membrane is efficient and
independent of microsolute concentration or size.
P ro t o c o l s f o r P ro t e in s C o n ce n t r a t io n, D e s a l t i n g a n d Bu f f e r E xch a n g e
Millipores Amicon Ultra-4 and -15 centrifugal
filters are designed for high speed with high
recovery. The devices incorporate low-binding
Ultracel regenerated cellulose ultrafiltration mem-
brane for sample concentration and purification of
solutions containing dilute or purified protein solutes,
antigens, antibodies, enzymes, or microorganisms.
Their speed and excellent recovery make them ideal
for desalting and buffer exchange applications.
One of the most common applications for Amicon
Ultra devices is concentration and desalting of
column fractions during protein purification by
various chromatography methods. Two examples
below demonstrate the use of Amicon Ultra devices
for high protein and enzymatic activity recovery.
34
Method
1. Select the device with the appropriate NMWL
and volume for the application.
2. Add the sample to the reservoir of the
centrifugal device.
3. If the sample is smaller than the maximum
volume, it can be diluted up to the maximum
volume before the first centrifugation step.
This will help increase the salt removal.
4. Centrifuge at the specified g-force for the
recommended amount of time.
5. Remove the initial filtrate from the filtrate tube
and set aside.
6. Add enough buffer or water to the device to
bring the sample volume up to 4 or 15 mL.
7. Centrifuge again.
8. Set aside the filtrate.
9. Recover the concentrated, de-salted sample.
NOTE: Both of the filtrates should be retained until
the concentrated sample has been analyzed.
Results
The transfer of salts across a membrane filter is
independent of sample concentration or size. There
is no change in the composition of the buffer when
desalting using ultrafiltration. For example, a solution
containing 500 mM salt still contains that concen-
tration after the initial centrifugation. Adding another
volume of salt-free buffer or water to the retentate
and centrifuging again will reduce the salt concen-
tration. This process, known as diafiltration, can
be repeated to achieve maximum salt removal.
Diafiltration can also be used when it is desirable to
have the sample in a different buffer. The sample is
concentrated and then repeatedly diluted with the
desired buffer and concentrated again.
 As the results show in Tables 1 and 2, the Concentration of Indoleamine 2,3-Dioxygenase
efficient design of the Millipore devices allowed
Courtesy of Eduardo Vottero,
>90% of the salt to be removed during the first
University of British Columbia
centrifugation step. Typically, only one subsequent
Indoleamine 2,3-dioxygenase (IDO; MW 48,000)
centrifugation step was needed to increase the
is a heme-containing enzyme that is the first and
typical salt removal to 99% with >90% recovery
rate-limiting enzyme in human tryptophan metabo-
of the sample.
lism. IDO processes 98% of the total tryptophan
Protein purification by chromatography usually
available in the human body and is critical in
involves the collection of multiple column fractions,
suppression of immunoresponse by blocking
with only some of those fractions containing the
T-lymphocyte proliferation locally [Swanson et al,
protein of interest. After the fractions are combined, a
Am J Respir Cell Mol Biol [manuscript in prepara-
protein concentration step is often required for protein
tion] (2003); Sarkhosh et al, J Cell Biochem 90,
storage, or concentration with buffer ex-change may
206 (2003); Mellor et al, J Immunol 171, (2003)].
be needed for downstream separations.

Table 1. Removal of sodium chloride and recovery of protein with Amicon Ultra-15 and Ultra-4 devices
Cytochrome c Cytochrome c BSA BSA IgG
0.25 mg/mL 0.25 mg/mL 1 mg /mL 1 mg/mL 1 mg/mL
NMWL 5 kDa 10 kDa 30 kDa 50 kDa 100 kDa
% Protein % NaCl % Protein % NaCl % Protein % NaCl % Protein % NaCl % Protein % NaCl
Spin

P ro t o c o l s f o r P ro t e in s C o n ce n t r a t io n, D e s a l t i n g a n d Bu f f e r E xch a n g e
Recovery Removal Recovery Removal Recovery Removal Recovery Removal Recovery Removal
1 94.5 97.2 97.3 97.9 96.0 98.2 98.9 97.1 99.9 97.7
2 92.6 99.9 95.6 99.9 94.4 99.9 92.4 99.9 97.1 99.5
Three Amicon Ultra-15 devices of each cut-off were tested with 15 mL of solute. 500 mM NaCl was added to each solution. Each spin
was performed at 4000 x g for 30 minutes. After the first spin, the retentate was brought up to 15 mL with ultrapure water from a Milli-Q
(Millipore) system. OD readings were taken at 410 nm for Cytochrome c and 280 nm for BSA and IgG.

Table 2. Removal of riboflavin and

recovery of IgG with Microcon filter
device with Ultracel membrane
Spin % NaCI % IgG
Number Remaining Recovered
1 9 95
2 2 93
3 <1 93
4 <1 92
500 L of a 50:50 mixture of riboflavin and IgG were
spun in a Microcon 3K NMWL device at 12,000 x g
for 75 minutes at room temperature in 55 angle rotor.
After the initial spin, the retentate was twice diluted
with 500 L of PBS and spun again. After each spin,
concentration of riboflavin and IgG in the filtrate and
retentate were monitored.

35
 Recombinant IDO was expressed in E. coli BL21
(DE3) cells utilizing the pET 28a (+) vector system.
In this system, a hexahistidyl tag was fused to full-
size IDO at the N-terminus with a spacer sequence
and a thrombin cleavage site. The protein was
purified by conventional His-tag purification
methods and eluted with imidazole. The histidine
tag was removed by thrombin cleavage. Final
purification was done by gel filtration chromatogra-
phy G-75. Amicon Ultra-15 centrifugal devices
were used to concentrate the IDO fractions from
an initial concentration of ~0.5 mg/mL to a
final concentration of 10 mg/mL. Samples were
analyzed by SDS-PAGE using a 12.5% polyacryl-
amide gel (Figure 1). In addition, it was shown that
no IDO activity loss was observed after concentra-
tion using an Amicon Ultra device.
Concentration of PKR and Buffer Exchange
Courtesy of Peter A. Lemaire and
Dr. James Cole, University of Connecticut
P ro t o c o l s f o r P ro t e in s C o n ce n t r a t io n, D e s a l t i n g a n d Bu f f e r E xch a n g e
developed for PKR, and PKR has been purified using
three chromatography steps on Agarose-Heparin,
Agarose-Poly (I), Poly (C) and Sephacryl S-200 gel
filtration columns. After the last step, PKR-containing
column fractions were pooled and concentrated
using Amicon Ultra-15 30K NMWL devices. The
concentration step was necessary for long-term
protein storage. Table 3 shows the protein recovery
results obtained after four concentrations. Over 90%
recovery was obtained and no protein loss to the
filtrate was observed.
Amicon Ultra-15 30K NMWL devices were also
used for exchanging buffer for PKR autophosphory-
lation (activation) assay. 200 L of 6.301 mg/mL
PKR in Protein Storage buffer (20 mM HEPES,
1 M NaCl, 10 mM b-mercaptoethanol, 0.1 mM
EDTA, 10% glycerol, pH 7.5) was diluted to 15 mL
with Phosphorylation Buffer (20 mM HEPES,
50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 1 mM
DTT, pH 7.5) and re-concentrated three times using

Human protein kinase R (PKR) is one of the major

proteins induced by interferon as part of the host Figure 1.
defense against viral infection14. PKR is synthesized
in a latent form and is activated by autophosphory-
lation induced upon binding dsRNA. Once
phosphorylated, active PKR phosphorylates the
40 kDa
eukaryotic translation initiation factor elF2a leading
to a block in protein synthesis in virally infected cells.
35 kDa
PKR has been implicated as a participant in various
signal transduction pathways associated with cellular
processes including transcription79, differentiation10,
apoptosis11, splicing14 and transformation5,6.
However, difficulties in purifying PKR in large
SDS-PAGE of purified indoleamine 2,3-dioxygenase
amounts has limited rigorous biophysical character-
before and after concentration using Amicon Ultra-15
ization of the mechanisms of PKR activation. A high- centrifugal devices.
yield prokaryotic expression system has been

Table 3. PKR concentration results

Spin 1 Spin 2 Spin 3 Spin 4
Starting Volume (mL) 15 15 15 15
Starting Concentration (mg/mL) 0.229 0.229 0.169 0.169
Volume of Retentate (L) 590 490 203 225
Total Amount of PKR (mg)
Before UF 3.4350 3.4350 2.5320 2.5320
After UF 3.1005 3.1548 2.4081 2.3983
Filtrate 0.0000 0.0000 0.0000 0.0000
% Recovery 90.26 91.84 95.11 94.72

36
Amicon Ultra devices at 3000 x g for 20 minutes at
4 C. The filtrates from the three steps were pooled
and the total amount of protein in all samples was
determined by UV absorption A280.
Protein activity was tested by autophosphoryla-
tion assay. The protein in the storage buffer was
supplemented with 5 mM MgCl2 and the samples
were allowed to undergo autophosphorylation at
30 C for 20 minutes in the presence of 3 mM ATP
and 3 Ci [g32P] ATP. The activity was determined
by autoradiography and quantified by liquid
scintillation counting. As shown in Figure 2, the
activity of PKR when no buffer exchange was only
6% of that when buffer exchange step was per-
formed. Hence, the UF successfully exchanged the
buffer while maintaining the activity of the protein.
References
1. Samuel CE. Antiviral actions of interferon.
Interferon-regulated cellular proteins and
their surprisingly selective antiviral activities.
Virology 1991;183(1):111.
2. Hovanessian AG. The double stranded RNA-
activated protein kinase induced by interferon:
dsRNA-PK. J Interferon Res 1989;9(6):641 7.
3. Lebleu B, et al. Interferon, double-stranded
RNA, and protein phosphorylation. Proc Natl
Acad Sci USA 1976;73(9):310711.
4. Samuel CE. Mechanism of interferon action:
phosphorylation of protein synthesis initiation
factor eIF-2 in interferon-treated human cells by
a ribosome-associated kinase processing site
specificity similar to hemin-regulated rabbit
reticulocyte kinase. Proc Natl Acad Sci USA
1979;76(2):6004.
5. Koromilas AE, et al. Malignant transformation
by a mutant of the IFN-inducible dsRNA-
dependent protein kinase. Science 1992;
257:16859.
6. Meurs E, et al. Tumor supressor function of
interferon-induced double-stranded RNA
activated protein kinase. Proc Natl Acad
Sci USA 1993;90:2326.
7. Wong AH, et al. Physical association between
STAT1 and the interferon-inducible protein
kinase PKR and implications for interferon and
double-stranded RNA signaling pathways.
EMBO J, 1997;16(6):1291304.
Figure 2.
100
80
Relative Activity of PKR
60
40
20
0
No Buffer Buffer
Exchange Exchange
Comparison of PKR autophosphorylation with buffer
exchange by ultrafiltration and without buffer
exchange.
8. Cuddihy AR, et al. Double-stranded-RNA-
activated protein kinase PKR enhances tran-
scriptional activation by tumor suppressor p53.
P ro t o c o l s f o r P ro t e in s C o n ce n t r a t io n, D e s a l t i n g a n d Bu f f e r E xch a n g e
Mol Cell Biol 1999;19(4):247584.
9. Demarchi F, Gutierrez MI, Giacca M. Human
immunodeficiency virus type 1 Tat protein
activates transcription factor NF-kappaB
through the cellular interferon-inducible, double-
stranded RNA-dependent protein kinase, PKR.
J Virol 1999; 73(8):70806.
10. Petryshyn R, et al. Effect of interferon on protein
translation during growth stages of 3T3 cells.
Arch Biochem Biophys 1996;326(2):2907.
11. Barber GN. Host defense, viruses and apopto-
sis. Cell Death Differ 2001;8(2):11326.
12. Tan SL, Katze MG. The emerging role of the
interferon-induced PKR protein kinase as a
apoptotic effector: A new face of death?
J Interferon Cytokine Res 1999;19:54354.
13. Balachandran S, et al. Activation of the
dsRNA-dependent protein kinase, PKR, induces
apoptosis through FADD-mediated death
signaling. EMBO J, 1998;17(23):6888902.
14. Osman F, et al. A cis-acting element in the 3-
untranslated region of human TNF-alpha mRNA
renders splicing dependent on the activation of
protein kinase PKR. Genes Dev 1999;13(24):
328093.
37
 Detergent Removal with Microcon
and Amicon Ultra Centrifugal Filters
Introduction Method and Results
Microcon and Amicon Ultra centrifugal filters are In a series of studies, Millipore researchers used
efficient laboratory tools for removing small mol- Total Organic Carbon (TOC) analysis to measure
ecules from solutions of proteins or nucleic acids. detergent removal using either Microcon or Amicon
Often, the molecule to be removed is one of a Ultra concentrators after a single centrifugation spin
number of commonly used detergents or protein (note that complete detergent removal generally
solubilizing agents. The chemical nature of most requires 35 spins). As the results in the Tables 1
detergents allows for micelle formation above a and 2 indicate, detergent removal depends both
critical concentration limit (Critical Micelle on the original detergent concentration and
Concentration, CMC). Micelle formation results in the NMWL of the centrifugal units. All measure-
aggregation of the detergent and leads to gross ments shown were made using detergent/
changes in molecular structure. This affects the distilled water solutions.
amount of the detergent that can be removed from
NOTE: Temperature, the presence of salts in
a solution by centrifugal devices with specific
the solution, and/or macromolecule/detergent
nominal molecular weight limit (NMWL)
interactions may lower the CMC for a particular
membranes.
detergent. Use the tables only as general guidelines
For example, the monomer of Triton X-100
in assessing the efficiency of detergent removal with
has a molecular weight of 500650 daltons.
the Microcon and Amicon Ultra devices.
Triton X-100 should pass readily through the
10,000 NMWL membrane in an Amicon Ultra
device. However, at concentrations above 0.01%
(0.2 mM), Triton X-100 forms micelles composed
of approximately 140 monomeric units. During
P ro t o c o l s f o r P ro t e in s D ete r g e n t Re m ova l

ultrafiltration, the micelles behave like 70,000

90,000 dalton globular proteins. As a result,
more than 90% of Triton is retained by the ultra-
filtration membrane. Therefore, above the CMC
of Triton X-100, an Amicon Ultra-4 100K NMWL
concentrator would be required to remove the
detergent effectively.

38
Table 1. Percent detergent removal after one spin with Table 2. Percent detergent removal
Microcon centrifugal devices after one spin with Amicon Ultra-4
centrifugal devices
NMWL
Detergent (%) 3 kDa 10 kDa 30 kDa 50 kDa 100 kDa NMWL
SDS Detergent (%) 10 kDa 30 kDa
0.001 >90% >90% >90% >90% >90% SDS
0.1 >90% >90% >90% >90% >90% 0.1 74% 75%
1 4089% 4089% 4089% 4089% 4089% 1 13% 11%
5 <40% <40% <40% <40% <40% 5 0.5% 2.0%
NaDeoxycholate Tween-20
0.1 >90% >90% >90% >90% >90% 0.1 47% 69%
1 >90% >90% >90% >90% >90% 1 36% 42%
5 4089% 4089% 4089% >90% >90% 5 39% 47%
CAPS Triton X-100
5 >90% >90% >90% >90% >90% 0.10 40% 70%
CPCl 1 1 26% 37%
0.01 >90% >90% >90% >90% >90% 5 37% 39%
0.1 4089% 4089% 4089% 4089% 4089% CHAPS
1 <40% <40% <40% <40% <40% 0.1 96% 81%
5 <40% <40% <40% <40% <40% 1 62% 96%
TDMABr2 5 36% 80%
0.1 >90% >90% >90% >90% >90%
1 <40% <40% <40% 4089% <90%
5 <40% <40% <40% <40% <90%
Digitonin
0.01 >90% >90% >90% >90% >90%
0.1 4089% 4089% 4089% 4089% 4089%
1 <40% <40% <40% <40% <40%
Tween-20
0.01 <40% <40% 4089% 4089% >90%
0.1 <40% <40% <40% 4089% >90%

P ro t o c o l s f o r P ro t e in s D ete r g e n t Re m ova l
1 <40% <40% <40% <40% 4089%
5 <40% <40% <40% <40% <40%
Triton X-100
0.01 4089% 4089% 4089% 4089% >90%
0.1 <40% <40% <40% <40% >90%
1 <40% <40% <40% <40% 4089%
5 <40% <40% <40% <40% <40%
CHAPS
0.1 >90% >90% >90% >90% >90%
1 4089% 4089% >90% >90% >90%
5 <40% <40% 4089% >90% >90%
1
Cetylpyridinium chloride
2
Tetradecyltrimethylammonium bromide

39
 Two-Dimensional Electrophoresis
Sample Preparation with
Amicon Ultra Centrifugal Filters
Two-dimensional electrophoresis (2DE) is one of the
most commonly used methods in proteome analysis.
Briefly, proteins are separated by their isoelectric
point (first dimension separation) followed by SDS-
PAGE separation by molecular weight (second
dimension separation). Although it is a powerful
method for simultaneously displaying hundreds
of proteins, 2DE presents a challenge for sample
preparation. Salts and ionic detergents are common
chemicals contaminating biological samples that
are not compatible with 2DE separation; however
P ro t o c o l s f o r P ro t e in s Tw o - D i m e n s io n a l E l e c t ro p h o re s i s S a m p l e P re p a r a t io n
Usually protein concentration is achieved by
protein precipitation with acetone or TCA. The
disadvantage of protein precipitation is that some
of the proteins become insoluble and can not be
resolubilized in IPG buffer. Another disadvantage is
that many salts become insoluble in acetone and
precipitate along with the proteins.
Ultrafiltration (UF) can achieve protein concentra-
tion and desalting in one step. Figures 1 and 2
present two examples of protein preparation for
2DE by acetone precipitation and ultrafiltration.

they are often required to solubilize proteins from
cells and tissues. The high concentration of salts
combined with the relatively low protein content
make samples completely unsuitable for isoelectric
focusing.
Both examples demonstrate that UF provides more
efficient salt removal and allows better separations
and improved resolution of protein spots in
two-dimensional electrophoresis.

Figure 1.

Two-dimensional electrophoresis of yeast cell lysate prepared by acetone precipitation (left) and ultrafiltration (right).
Sacharomyces cerevisiae strain s288c was grown to log phase. The cells were pelleted and resuspended in Cellular
and Organelle Membrane Solubilizing Reagent from the ProteoPrep kit (Sigma) and lysed by sonication. Cellular
debris was removed by centrifugation and the supernatants were reduced and alkylated with 5 mM tributylphosphine
and 10 mM acrylamide. Lysates were acetone-precipitated to remove residual Tris and alkylating reagent or were
filtered through Amicon Ultra-4 10K NMWL devices. Samples were redissolved in ProteomIQ Resuspension Reagent
(Proteome Systems), focused in broad range (pH 310) immobilized pH gradients (IPGs) on the IsoelectrIQ IEF
instrument. Second dimension gels were 615% polyacrylamide gradient GelChips (Proteome Systems) run on
an ElectrophoretIQ 2D instrument (Proteome Systems). Data courtesy of Dr. G. Smejkal, Proteome Systems, Inc.,
Woburn, MA, USA.

40
Figure 2.

P ro t o c o l s f o r P ro t e in s Tw o - D i m e n s io n a l E l e c t ro p h o re s i s S a m p l e P re p a r a t io n
Two-dimensional electrophoresis of endothelial cell lysates prepared by acetone precipitation (left) and ultrafiltration
with Amicon Ultra devices (right). Whole cell lysates of endothelial cells were prepared in 7 M urea, 2 M thiourea,
4% CHAPS, 10 mM DTT, 20 mM Tris buffer. The samples were very dilute and presumably contained DNA fragments,
salts and lipids. 300 mg of the total protein was either acetone-precipitated or desalted in Amicon Ultra devices.
After acetone precipitation with four volumes of cold acetone, samples were incubated overnight at 20 C,
precipitated by centrifugation, rinsed with cold acetone, and pelleted again. The resulting pellet was resuspended
in rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 10 mM DTT, 20 mM Tris). Alternatively, samples were
filtered through Amicon Ultra-4 10K NMWL devices after being mixed with nine volumes of 20 mM Tris HCl, pH 7.
The sample was concentrated to approximately 40 L and adjusted to 350 L with rehydration buffer. The proteins
were separated by IEF in 18 cm IPG Drystrips, pH 310 (GE Healthcare). A second dimension separation was
performed in 10% SDS-PAGE gel. The gels were silver stained and scanned. Data courtesy of Dr. Leonid Kryazhev,
Genome Quebec, Montreal, Canada.

41
 Purification of Serum Peptides for
Biomarker Research with Amicon Ultra
Centrifugal Filters
Introduction
A biomarker can be defined as a molecule that
indicates an alteration in physiology. Biomarkers
play an essential role in the drug discovery and
development process. They provide powerful clues
to genetic susceptibility, disease progression, and
predisposition, as well as drug response and the
physiological and metabolic profiles of diseases
and drug responses. Biomarkers can also provide
valuable diagnostic and prognostic information that
P ro t o c o l s f o r P ro t e in s P u rif i c a t io n of S e ru m Pe p t i d e s f o r Bio m a rke r Re s e a rch
can facilitate personalized medicine. Peptide and
protein patterns have been linked to ovarian cancer,
breast cancer, prostate cancer and astrocytoma15.
Most diagnostic tests are based on blood or
urine analysis5. Serum is a key source of putative
protein biomarkers, and, by its nature, can elucidate
organ-confined events. Mass spectrometry, coupled
with bioinformatics, is capable of distinguishing
between serum protein pattern signatures in late-
stage and early-stage ovarian cancer patients6.
One of the major impediments to the discovery of
new biomarkers is the presence of salts, proteins,
and lipids in plasma or serum that makes it difficult to
detect and analyze peptides by mass spectrometry.
When untreated serum is spotted onto a MALDI-TOF
plate, it does not produce any usable signal in mass
spectrometry. Multiple protocols have been devel-
oped to extract and enrich peptides from tissues and
body fluids, such as batch reversed phase chroma-
tography over C18 resin and extraction with 0.1%
trifluoroacetic acid (TFA) or 50% acetonitrile to
selectively precipitate large proteins while enhancing
the solubility of smaller proteins and peptides.
Ultrafiltration has previously been reported as a
sample preparation tool to prepare low molecular
weight fractions for biomarker analysis710. In this
study we show that ultrafiltration in combination with
solid phase extraction (SPE) on C18 resin can be a
convenient and efficient method for serum peptide
purification. The approach provides more peptides
for mass spectrometry analysis than the acetonitrile
precipitation method.
42
Methods
Preparation of Serum Peptides by UF and SPE
One milliliter of human serum, with or without
acetonitrile, was filtered using Amicon Ultra-4
10K NMWL centrifugal devices. The devices were
centrifuged in a swinging bucket rotor for 15 to
30 minutes at 3000 x g. Ten microliters of the
filtrate was acidified with 5 L of 1% TFA, concen-
trated with ZipTipC18 pipette tips. Co-elution was
performed directly onto a MALDI target with 2 L of
-cyano-4-hydroxycinnamic acid matrix (5 mg/mL
in 50% acetonitrile, 0.1% TFA). If acetonitrile was
added to the serum prior to the filtration, the samples
were briefly evaporated in a Speed Vac centrifuge
to remove solvent before ZipTip purification.
Peptide Analysis by Mass Spectrometry
Peptide-containing ultrafiltrates from cell lysates
or human serum were acidified with 1% TFA and
concentrated on ZipTipC18 or ZipTipSCX pipette tips
following the procedure outlined in the user guide.
All samples were overlaid with 1 L of -cyano-
4-hydroxycinnamic acid matrix (5 mg/mL in
50% acetonitrile, 0.1% TFA) and analyzed on
Voyager-DE Workstation (Applied Biosystems)
in linear mode.
Preparation of Serum Peptides by
Acetonitrile Precipitation
Acetonitrile was added to human serum at a
1:1 ratio (v:v), and samples were centrifuged to
precipitate larger proteins. The supernatant was
dried in a Speed-Vac centrifuge, resuspended in
0.1% TFA, and then desalted and concentrated with
ZipTipC18 pipette tips. Co-elution was performed
directly onto a MALDI target with 2 L of -cyano-
4-hydroxycinnamic acid matrix (5 mg/mL in
50% acetonitrile, 0.1% TFA).
Results One of the common methods for serum peptide
preparation for mass spectrometry is acetonitrile
While human serum contains numerous peptides
fractionation, where the addition of 5070%
and small proteins, they are not accessible by direct
acetonitrile precipitates larger proteins, while the
mass spectrometry analysis. Even after reverse
peptides stay soluble in the supernatant. Another
phase concentration and desalting, only a few
way to produce relatively protein-free filtrates is
peptides are detectable in the mass spectrum
ultrafiltration. Figure 2 presents the MALDI-TOF
(data not shown). This can be explained by the
spectra of (A) straight rat serum; (B) serum superna-
high concentration of proteins and lipids competing
tant after 50% acetonitrile precipitation; and (C)
with the peptides to bind to the resin (Figure 1A).

Figure 1.

A B C

P ro t o c o l s f o r P ro t e in s P u rif i c a t io n of S e ru m Pe p t i d e s f o r Bio m a rke r Re s e a rch

MALDI-TOF spectra of (A) unprocessed rat serum; (B) serum peptides in 50% acetonitrile supernatant; and (C) serum ultrafiltrate
processed with Amicon Ultra-4 30K NMWL centrifugal device.

Figure 2.

A B

C D

MALDI-TOF spectra of rat serum peptides after concentration and desalting by reversed
phase chromatography: (A) rat serum processed with ZipTipC18 pipette tip; (B) serum
supernatant after 50% acetonitrile precipitation processed with ZipTipC18 pipette tip;
(C) 30K serum ultrafiltrate processed with ZipTipC18 pipette tip; and (D) the same as (C)
but 20% acetonitrile was added to the serum prior to ultrafiltration.

43
 serum ultrafiltrate processed with an Amicon Ultra-4
30K NMWL device. We observed higher quality
spectra and an increased number of MALDI-TOF
detected peptides if the serum was ultrafiltered.
Further improvement of spectra can be achieved
by reverse phase chromatography, which concen-
trates and desalts the peptides. The ZipTipC18
pipette tip is a convenient and efficient tool for
micro-scale sample preparation prior to mass
spectrometry. Figure 2 shows the MALDI-TOF
spectra of rat serum peptides prepared with
ZipTipC18 pipette tip out of (A) straight serum,
(B) 50% acetonitrile supernatant and (C) serum
processed by ultrafiltration. The last method
provided stronger MALDI-TOF signal, higher
signal-to-noise ratio and double the number of
detected peptides.
The addition of 5 to 10 % acetonitrile to serum
P ro t o c o l s f o r P ro t e in s P u rif i c a t io n of S e ru m Pe p t i d e s f o r Bio m a rke r Re s e a rch
and plasma samples (data not shown) prior to
ultrafiltration was shown to improve the detection
of serum peptides, the MALDI spectrum and the
overall signal intensity.
Conclusion
For the analysis of serum peptides, complexity
reduction by eliminating higher molecular weight
proteins is critical for high resolution mass spec-
trometry. Efficient separation of peptides from the
majority of proteins and salts can be achieved by
sample ultrafiltration. We have shown the effective
use of Amicon Ultra-4 30K NMWL centrifugal
devices for the preparation of peptides. Other
molecular weight cut-offs can be utilized depending
on the desired range of peptides. The method can
be used directly in combination with ZipTipC18
pipette tips for peptide identification by MS/MS
or as a first step prior to further surface-mediated
enrichment using SELDI-TOF methods. These sample
preparation protocols may also be applicable to
other low molecular weight markers such as drugs
and metabolites. Peptide purification using ultrafiltra-
tion to de-proteinize followed by SPE to de-salt
serum and plasma samples (data not shown) was
easily adapted to the 96-well format allowing large
numbers of samples to be simultaneously prepared.
44
References
1. Ardekani AM, Liotta LA, Petricoin III. Expert Rev
Mol Diagn 2002;2:12.
2. Carter D, Douglass JF, Cornellison CD, Retter
MW, Johnson JC, Bennington AA, Fleming TP,
Reed SG, Houghton RL, Diamond TS, Vedvick
TS. Biochemistry 2002;41:6714.
3. Wellmann A, Wollscheid V, Lu H, Ma ZL,
Albers P, Schutze K, Rohde V, Behrens P,
Dreschers S, Ko Y, Wernert N. Int J Mol Med
2002;9:341.
4. Petricoin EF, Ardekani AM, Hitt BA, Levine PJ,
Fusaro VA, Steiberg SM, Mills GB, Simone C,
Fishman DA, Kohn EC, Liotta LA. Lancet 2002;
359:572.
5. Bischoff R, Luider TM. J Chrom B 2004;803:
2740.
6. Stevens EV, Liotta LA, Kohn EC. J Gynecol
Cancer 2003;13:1339.
7. Schulz-Knappe P, Schrader M, Standker L,
Richter R, Hess R, Jurgens M, Forssmann W-G.
J Chromatogr A 1997;776:125132.
8. Basso D, Valerio A, Seraglia R, Mazzza S,
Piva MG, Greco E, Fogar P, Gall N,
Pedrazzoli S, Tiengo A, and Plebani M.
Pancreas 2002;24:814.
9. Prazeres S, Santos MA, Ferreira HG, Sobrinho
LG. Clin Endocrinol (Oxf) 2003;58:68690.
10. Tirumalai RS, Chan KC, Prieto DA, Issaq HJ,
Conrads TP, Veenstra TD, Mol Cell Proteomics
2003;10:1096103.
11. Chernokalskaya E, Gutierrez S, Pitt AM,
Leonard J. Electrophoresis 2004;25:
24612468.
12. Millipore Case Study PR0001EN00.
Automating Multiplexed Cytokine Assays
manuscript in preparation.
Rapid Antibody Concentration
with Amicon Ultra Centrifugal Filters
Ultrafiltration offers a fast and convenient way to
concentrate antibodies purified from serum, ascites Figure 1.
fluid, or hybridoma supernatants. The traditional 40 12
purification protocol for immunoglobulins includes

IgG Concentration (mg/mL)

PROSEP-A
10
affinity binding to Protein A or G chromatography 30 PROSEP-G

IgG Volume (mL)

media, washing unbound proteins, and eluting with 8

a low pH buffer. Subsequently, purified antibodies 20 6

are often too dilute for their intended purpose or for
4
long-term storage. In addition, harsh elution 10
conditions sometimes require buffer exchange to 2
preserve protein activity. 0 0
Dialysis is often used for antibody concentration 0 3 10 15 20

and buffer exchange. However, ultrafiltration Centrifugation Time (min)

provides a quick, alternative method to concentrate Concentration of rabbit IgG with Amicon Ultra-15 devices. The IgGs were
and diafilter immunoglobulins with up to 99% purified using Montage PROSEP-A or PROSEP-G Antibody Purification Kits.
The lines show IgG volume reduction, while the bars show a proportional
recovery and one-step salt removal (see
increase in IgG concentration after 20 minutes of centrifugation time.
Concentration, Desalting, and Buffer Exchange with
Amicon Ultra or Microcon Centrifugal Filters, page
34).
To demonstrate the suitability of Amicon Ultra Figure 2.

P ro t o c o l s f o r P ro t e in s Ra p i d A n t i b o d y C o n ce n t r a t io n
devices for concentrating purified IgG, rabbit serum 1 2 3 4 5
SDS-PAGE gel of purified kDa
was processed with Montage Purification Kits with
rabbit IgG before and 116
PROSEP-A and PROSEP-G Antibody (Millipore) after concentration with
and then concentrated with Amicon Ultra-15 30K Amicon Ultra-15 devices.
66
NMWL devices. Figure 1 shows a decrease in the Lane 1: MW standards 55
Heavy
retentate volume proportional to the increase in Lanes 2, 3: PROSEP-A-purified IgG Chain

antibody concentration. A twenty-minute centrifuga- before concentration 36

(lane 2) and after
tion resulted in >95% recovery of immunoglobulins. concentration (lane 3) Light
21 Chain
Figure 2 shows an SDS-PAGE gel of purified rabbit Lanes 4, 5: PROSEP-G-purified IgG
before concentration 14
IgGs before and after ultrafiltration.
(lane 4) and after
concentration (lane 5)
Protein Load: 5 L in lanes 2 and 4;
1 L in lanes 3 and 5

45
 Affinity Purification with
Ultrafree-MC Centrifugal Filters
Introduction
Affinity interaction chromatography is often the
single most effective step in any protein purification
procedure. Up to 95% purity can be achieved in
one step, depending on the nature of the interaction
and the starting composition of the protein solution.
Well known examples of highly specific affinity
interactions include antibodies and protein A/G;
multiple histidine tags and nickel; streptavidin and
biotin; antibodies and antigens; and many others.
Ultrafree-MC centrifugal filter unit
Less specific interactions are also used for enrich-
ment or depletion protocols, including albumin
depletion on cibacron blue resin, glycoprotein Another alternative for small-scale purification
enrichment on concavalin A resin, and capture are centrifugal devices with microporous membrane,
of nucleic acid-binding proteins on heparin resin. such as Ultrafree-MC centrifugal devices. The
Affinity chromatography is often employed in sample can be added to the filter basket and mixed
the small-scale batch mode as a quick method for for the needed residence time and then centrifuged.
microgram-scale protein purification. The typical The process removes the interstitial liquid but does
protocol involves: not dehydrate the beads. Washing and elution
1. Pipetting a small volume of affinity resin into can also be performed in a similar manner and
a microfuge tube that contains the sample are more effective due to the efficient removal of
2. Vortexing the tube for a few minutes buffer and/or eluant.
Ultrafree-MC centrifugal filter units with micropo-
P ro t o c o l s f o r P ro t e in s A f f i n i t y P u rif ic a t io n

3. Centrifuging the resin to the bottom

4. Pipetting off the supernatant
5. Washing a few times (using steps 2 and 3)
6. Eluting with a small amount of eluant
Although the method is relatively simple, care
must be taken not to remove the chromatography
resin when pipetting off the supernatant.
Pre-packed mini-spin columns are a convenient
tool for small-scale protein purification. Operated
by centrifugation, they usually complete the whole
procedure in less than an hour.
rous membrane come with low protein-binding
Durapore PVDF membrane in five different pore
sizes from 0.1 to 5.0 m. Affinity resin can be
loaded into the filter basket and the device used
as a home-made mini-spin column.
We show the applicability of the device for
purification of rabbit IgG on PROSEP-A resin and
His-tagged C-RP protein on three different commer-
cial metal-chelate resins.

46
Materials
Ultrafree-MC 0.45 m centrifugal devices
(Millipore cat. no. UFC3 0HV 00)
PROSEP-A high capacity resin
(Millipore cat. no. 1131 118 26)
Rabbit serum Gibco (Invitrogen lot no.
1132782)
Micro-centrifuge Biofuge Pico
(Heraeus instruments)
Jouan CR1822 fixed angle rotor centrifuge
Xcell SureLock Mini-cell vertical electrophoresis
system (Invitrogen cat. no. EI0001)
NuPage NOVEX Bis-Tris 4 12%, 1 mm thick,
15 well SDS gels, (Invitrogen cat. no. NP0323)
NuPage Sample Reducing agent (10X)
(Invitrogen cat. no. NP009)
NuPage SDS Sample Buffer (4X)
(Invitrogen cat. no. NP007)
SimplyBlue SafeStain Coomassie G-250 stain
(Invitrogen cat. no. LC6060)
Method for IgG Purification
Solutions
PROSEP-A binding buffer A: 1.5 M Glycine/
NaOH, 3 M NaCl, pH 9.0
PROSEP-A elution buffer B2: 0.2 M Glycine/
HCl, pH 2.5
PROSEP-A neutralization buffer: 1 M Tris/HCl,
pH 9.0
Procedure
1. 200 mg of PROSEP-A media were placed in
Ultrafree-MC 0.45 m filter basket.
2. The columns were equilibrated with 400 L of
binding buffer A and centrifuged for 1 minute
at 100 x g.
3. 200 L of rabbit serum were diluted 1:1 with
binding buffer and the entire volume was loaded
into the spin column containing PROSEP-A resin.
4. Devices were placed on a shaker for 15 minutes
at room temperature and centrifuged at 100 x g
for 5 minutes. Flow-through was collected for
future analysis.
5. Three consecutive washes of 400 L each were
performed by adding 400 L of binding buffer A
and centrifuging at 2,000 x g for 2 minutes each.
6. 200 L of elution buffer B2 were added and
centrifuged for 2 minutes at 2,000 x g.
P ro t o c o l s f o r P ro t e in s A f f i n i t y P u rif ic a t io n
7. 26 L of neutralization buffer were added to
each collection tube. A second elution was
collected after repeating the same process one
more time.
47
 Method for His-tagged C-RP Purification
Solutions
Lysis buffer: 50 mM sodium phosphate, 300 mM
sodium chloride, 10 mM immidazole, pH 7
Binding buffer: 50 mM sodium phosphate,
300 mM sodium chloride, 10 mM immidazole,
pH 7
Wash buffer: 50 mM sodium phosphate, 300 mM
sodium chloride, 20 mM immidazole, pH 7
Elution buffer: 50 mM sodium phosphate,
300 mM sodium chloride, 250 mM immidazole,
pH 7
1 mg/mL Lysozyme stock
Benzonase
Procedure
1. Recombinant proteins were expressed in
Escherichia coli.
2. Cells were prepared at a 10X concentration
using lysis buffer. Lysozyme was added to a
concentration of 0.1 mg/mL. To reduce the
viscosity, benzonase was added to the lysate.
The lysates were clarified by centrifugation.
3. 200 L of the 50% resin slurry were added to
the Ultrafree-MC device and the residual fluid
was removed by centrifugation for 1 minute at
P ro t o c o l s f o r P ro t e in s A f f i n i t y P u rif ic a t io n
500 x g.
4. The resin was equilibrated with 500 L of
binding buffer and centrifuged for 2 minutes
at 500 x g.
48
5. 500 L of the clarified lysate were added to
the resin.
6. The His-tagged proteins were bound for
30 minutes with light agitation.
7. The lysate was removed by centrifugation at
500 x g for 10 minutes.
8. The resin was washed with 500 L of wash
buffer for 5 minutes with agitation. The wash
solution was removed by centrifugation for
5 minutes at 500 x g. This step was repeated
two more times.
9. 250 L of elution buffer were added to the
Ultrafree-MC device and mixed for 5 minutes.
Purified protein was recovered by centrifugation
at 500 x g for 1 minute.
Results
The results of purifying rabbit IgG using Ultrafree-
MC centrifugal devices are shown in Figure 1.
The device was challenged with approximately
14 mg of total protein, with an estimated IgG
content of 1.52 mg. The original serum, flow-
through, three washes and two eluted fractions
were analyzed by SDS-PAGE. The total amount of
purified IgG, as estimated by OD280, was 1.2 mg
and 1.1 mg on two devices processed in parallel.
The whole procedure was completed in less than
one hour. This method can be useful for monitoring
the titer of antigen-specific antibodies after immune
activation, or whenever small amounts of IgG need
to be purified.
 Figure 2 shows the results of His-tagged protein
purification in Ultrafree-MC devices using Ni-NTA Figure 1.
1 2 3 4 5 6 7 8
agarose and BD Talon resin. Two recombinant Rabbit IgG purification kDa
200
proteins were purified: C-RP, high-expressing protein on PROSEP-A resin in
of 26 kDa; and RT66, low-expressing protein of Ultrafree-MC devices. 116.3
97.4
66 kDa. Both purifications resulted in high purity Lane 1: Molecular 66.3
weight 55.4
proteins. The amount of proteins purified out of standards
500 L of lysate was 3235 g for C-RP protein Lane 2: Rabbit serum 36.5
and 8 g for RT66. Lane 3: Flow through 31.0

The data show that affinity batch purification Lanes 46: Three consec- 21.5
can be effectively performed on a small scale using utive washes
14.4
Lanes 7, 8: Eluted IgG
Ultrafree-MC devices loaded with resin. The
from two
6.0
process combines the high efficiency of batch devices
binding and washing with the handling conve-
nience of a mini-spin column. With minimal hands-
on time, the method provides flexibility of resin to
lysate ratio and binding conditions, independent of Figure 2.
centrifugation speed and rotor angle. This method 1 2 3 4 5 6 7
His-tagged protein kDa
200
is applicable to recombinant protein purification, purification using
antibody purification and immunoprecipitation. Ultrafree-MC devices. 116.3
97.4
Lane 1: Molecular 66.3 RT66
weight 55.4
standards
Lane 2: E. coli lysate 36.5
expressing 31.0
C-RP
C-RP protein
21.5
Lane 3: E. coli lysate
expressing 14.4
RT66 protein
Lanes 4, 5: Proteins 6.0
purified on

P ro t o c o l s f o r P ro t e in s A f f i n i t y P u rif ic a t io n
Ni-NTA resin
Lanes 6, 7: Proteins purified
on BD Talon resin

49
 Removal of Unincorporated Label
from Labeled Protein with Amicon Ultra
Centrifugal Filters
Introduction
Centrifugal devices containing ultrafiltration mem-
branes are ideal for the removal or exchange of
salts, sugars, nucleotides, non-aqueous solvents,
and other materials of low molecular weight. They
also serve to separate free from bound species.
For the removal of unincorporated label in protein
labeling applications, Millipore centrifugal devices
provide fast, convenient, high-recovery alternatives
to gel filtration. Sample dilution, often associated
P ro t o c o l s f o r P ro t e in s Re m ova l of Un i n co rp o r a te d L a b e l f ro m L a b e l e d P ro te i n
Results
Figure 1 shows the SDS-PAGE gel of FITC-labeled
BSA before and after each of four diafiltration cycles.
Unincorporated FITC is clearly visible in the starting
material and in the first filtrate. After only one cycle
of ultrafiltration, the majority of the free label is
removed. Subsequent cycles of filtration result in
BSA that is virtually free of unincorporated FITC.
Fluorescence measurement offers a more sensitive
method to monitor the ultrafiltration process. Figure

with gel filtration, is not a problem. A few rounds of 2 shows the change in fluorescence of filtrate and
diafiltration can efficiently remove unincorporated retentate during four cycles of FITC-BSA diafiltration.
label. Two examples below demonstrate the use The results indicate that almost 80% of free FITC can
of Millipore centrifugal ultrafiltration devices for be removed after the first ultrafiltration and three
removal of unreacted fluorescent and isotope labels. rounds result in 98% removal. This demonstrates
the viability of ultrafiltration as an alternative to gel
Method for Removal of FITC from FITC-BSA filtration for cleaning up protein labeling reactions.
1. Bovine serum albumin was labeled with Using an Amicon Ultra-4 device, each ultrafiltration
fluorescein-isothiocyanate (FITC); the free is accomplished in 1015 minutes and allows high
unincorporated label was not removed. recovery of target protein. While gel filtration results
in diluted protein fractions, ultrafiltration offers the
2. Two mL of FITC-labeled BSA solution at
additional advantage of concentrating protein while
0.5 mg/mL were loaded into two Amicon
removing unincorporated label.
Ultra-4 10K NMWL devices and centrifuged
at 3,000 x g for 10 minutes.
3. Retentates (about 50 L each) were re-diluted
to 2 mL with water and centrifuged again.
This step was repeated twice.
4. After each ultrafiltration, the retentate and
filtrate were analyzed by SDS-PAGE and their
fluorescence measured on a SpectraFLUOR
plate reader (Tecan) at excitation 485 nm and
emission 530 nm.
5. The SDS PAGE gel was scanned on a Storm
(GE Healthcare) fluorescence scanner.

50
Figure 1. Figure 2.
SDS-PAGE of FITC-labeled BSA 60,000

Lane 1: Starting material 50,000 Retentate

Lane 2: Retentate after first ultrafiltration Filtrate

(Aribitrary Units)
40,000

Fluorescence
Lane 3: Retentate after 2 rounds of ultrafiltration
Lane 4: Retentate after 3 rounds of ultrafiltration 30,000
Lane 5: Retentate after 4 rounds of ultrafiltration
20,000
Lane 6: Filtrate after first ultrafiltration
Lane 7: Filtrate after 2 rounds of ultrafiltration 10,000
Lane 8: Filtrate after 3 rounds of ultrafiltration
0
Starting Spin 1 Spin 2 Spin 3 Spin 4
material
Centrifugation Time (min)
FITC-BSA
Fluorescence measurements of the filtrates and retentates after each of four
cycles of FITC-labeled BSA ultrafiltration. Free label was transferred through
the 10,000 NMWL membrane while labeled BSA was retained and
concentrated. All retentates and filtrates were volume adjusted to 2 mL
prior to the measurement.

P ro t o c o l s f o r P ro t e in s Re m ova l of Un i n co rp o r a te d L a b e l f ro m L a b e l e d P ro te i n
FITC

1 2 3 4 5 6 7 8

51
 Rapid, Ultrafiltration-based Method for
Purification of Monoclonal Antibodies
Introduction desalted and exchanged into a biological buffer
using dialysis. The entire process typically requires
Monoclonal antibodies (MAb) continue to gain
several days to complete and can be particularly
importance as therapeutic and diagnostic agents
onerous if multiple MAbs are to be evaluated in
for many types of cancer. The process of screening
parallel. We describe here a new and simplified
hybridoma libraries for candidate MAbs is both time
method that minimizes the processing time to less
consuming and labor intensive. Once a hybridoma
than a day to obtain pure MAb.
cell line expressing a suitable MAb is established,
a bench-scale purification methodology (e.g., 50
Method
to 500 mL) must be developed to produce sufficient
The method involves clarification of the hybridoma
MAb for further characterization.
supernatant by microfiltration using a Stericup
A traditional method for purifying MAbs involves
vacuum filter cup, followed by concentration using
clarification of the hybridoma supernatant by
ultrafiltration1 with a Centricon Plus-70 device
centrifugation, followed by an ammonium sulfate
(see Table 1 and Figure 1 for method details).
precipitation to concentrate the MAbs. The precipi-
The MAb is further purified on protein A/G beads.
tate is then recovered by centrifugation, resolubi-
The purified MAb is desalted and buffer-exchanged
P ro t o c o l s f o r P ro t e in s Ra p i d P u rif ic a t io n of M o n o cl o n a l A n t i b o d ie s

lized and desalted using dialysis. After these steps,

using ultrafiltration.
the MAb is further purified using Protein A/G
affinity chromatography. The purified antibody is

Table 1. Comparison of traditional and ultrafiltration workflows

Workflow for Obtaining Concentrated Supernatant

Traditional Ultrafiltration
Ammonium Sulfate Precipitation Centrifugal Tangential Flow Filtration (TFF)
1. Start with 200 mL 1. Start with 200 mL 1. Start with 1000 mL
2. Weigh out ammonium sulfate 2. Add supernatant to 2. Add supernatant to Labscale
3. Add ammonium sulfate to Centricon Plus-70 Device TFF unit (30K MWCO)
clarified supernatant with (100K MWCO) 3. Apply pressure (40 psi) with
constant stirring (20-30 min). 3. Centrifuge (2030 min) constant stirring (~2 hrs)
Store at 4 C. 4. Invert spin to collect sample 4. Collect sample (5 min)
4. Centrifuge to recover pellet, (2 min)
resuspend pellet 5. Load on protein-G column
5. Prepare dialysis membrane
and check for integrity
6. Collect resuspended
precipitate in dialysis
tubing/device
7. Dialyze with three changes
of PBS (18-24 hrs)
8. Collect dialysate in
centrifuge tube(s) and
centrifuge (~30 min)
9. Load on protein-G column
Total time required ~24 hrs Total time required ~40 min Total time required ~2 hrs

52
Results
Figure 2.
This approach was successfully used to purify an
2 Abalone Commercial
anti c-myc antibody secreted by the hybridoma
clone 9E102 (Figure 1). The MAb purified by this
new method performed comparably to the commer-
cially purified MAb in downstream applications
such as western blotting and ELISA (Figure 2). The
data demonstrate that the new protocol is robust
and delivers MAb of a high purity and yield as
compared to the traditionally purified MAb. The Traditional UF-based

application of ultrafiltration to MAb purification

will be of considerable value to any researcher
interested in screening hybridoma libraries and
accelerating the purification of MAbs.

Figure 1. 0.7
C Tr UF
C: Clarified supernatant UF-based method
0.6
Tr: Supernatant precipitated Traditional method
OD @ 450 mm

0.5
with ammonium sulfate and Commercially available
dialyzed using 10K MWCO 0.4

P ro t o c o l s f o r P ro t e in s Ra p i d P u rif ic a t io n of M o n o cl o n a l A n t i b o d ie s
membrane (Spectrapor,
Rancho Dominguez, CA) 0.3
UF: Supernatant concentrated 0.2
by ultrafiltration on
Centricon Plus 70 device 0.1

0.0
0 10 100

Concentration of mAB (g/mL)

Upper panel shows activity comparison measured in a western blot

Conclusion
Our data show that the combination of microfiltra-
tion and ultrafiltration is a rapid method for MAb
purification and that MAb purified on Montage
centrifugal columns with Protein G media is compa-
rable to the commercially available MAb in purity,
activity and cost. The Ultrafiltration-based MAb
purification method, compared to the traditional
method, is faster (23 hours versus 2 days), easier
to use, and yields higher recoveries.
References
1. Saha K, Case R, Wong PK. J Immunol Methods
1992;151(1-2):307-308.
2. Evan GI, Lewis GK, Ramsay G, Bishop JM. Mol
Cell Biol 1985;5(12):3610-3616.
between MAb (derived from HEK 293) purified using ultrafiltration method
versus traditional method. Three-fold serial dilutions of HEK 293 cell
nuclear extracts were run on 4 to 12% NuPAGE gels and transferred to
Immobilon-P membrane (Millipore). Blots were probed with the indicated
primary antibodies and secondary anti-mouse alkaline phosphatase
conjugate (Sigma, St. Louis, MO). The antibodies were detected with
Immobilon Western AP substrate (Millipore) and imaged on a Kodak
scanner.
Bottom panel shows activity comparison measured in an ELISA
assay. HEK 293 cells were grown on 96-well plates. After fixation and
epitope-retrieval by heating for 10 minutes in a microwave, cells were
permeabilized with 1% saponin (Sigma) in PBS + 2% normal donkey serum
(Jackson Immunoresearch, West Grove, PA) and treated with serial dilutions
of the indicated purified MAbs. The cells were then washed and treated
with goat anti-mouse HRP-conjugate antibody (Sigma). The reactions were
developed using a SureBlue TMB HRP substrate (KPL, Gaithersburg, MD).
The readings were measured on a SpectraMax plate reader (Molecular
Devices, Sunnyvale, CA).

53
 A Simple Strategy for Protein
Enrichment Using Ultrafiltration
Introduction original concentration. These limitations can be
overcome by using centrifugal ultrafiltration devices
The human plasma proteome is a diverse universe
for enrichment.
of information and it will continue to be a vast
source of biomarker discovery and investigation.
Method
Ultrafiltration (UF) has been reported13 as a sample
This experiment demonstrates the use of ultrafiltration
preparation tool to prepare low molecular weight
devices to enrich both LMW and high molecular
(LMW, <10 kDa) fractions by reducing the complex-
weight (HMW) fractions from serum. Protein
ity of serum proteins prior to mass spectrometry for
separation was accomplished by serial filtration
biomarker analysis. Traditionally, enrichment of
through decreasing molecular weight cutoff
protein solutions on the basis of size has been
(MWCO) devices from 100K to 10K MWCO.
accomplished using gel filtration chromatography4,5.
This serial enrichment strategy enabled the com
However, gel filtration is laborious, limited by
partmentalization of proteins by size as well as an
sample size and time-consuming. Another conse-
increase in throughput as compared to directly
quence of gel filtration chromatography is the
enriching samples using a lower MWCO device.
significant dilution of the protein relative to the
P ro t o c o l s f o r P ro t e in s P ro te i n E n rich m e n t U s i n g U l t r af i l t r a t io n

Figure 1. Serial enrichment strategy Figure 2. Purification of Cytochrome c from a mixture

containing 10% fetal bovine serum (FBS) and the
Step 1: 100 kDa MWCO compartmentalization of protein fractions using
40 min at 1500 x g
Amicon Ultra devices

100 kDa Retentate 100 kDa Permeate M S R100 P100 R50 P50 R30 P30
(<100 kDa)

100 kDa
Step 2: 50 kDa MWCO
30 min at 1500 x g
50 kDa

50 kDa Retentate 30 kDa

50 kDa Permeate
(<50 kDa)
Cytochrome c Purified
(12.5 kDa) Cytochrome c

Step 3: 30 kDa MWCO

M: Marker R50: 50 kDa Rententate
30 min at 1500 x g
S: 10% FBS + 1 mg/mL Cytochrome c P50: 50 kDa Permeate
R100: 100 kDa Retentate R30: 30 kDa Retentate
30 kDa Retentate P100: 100 kDa Permeate P30: 30 kDa Permeate
30 kDa Permeate
(<30 kDa)
A serum-protein mixture spiked with purified Cytochrome c (12.5 kDa)
A complex mixture is centrifuged in a 100K device. was centrifuged in an Amicon Ultra 4 mL device using serially decreasing
The retentate is removed and saved for later analysis. MWCOs as described in Figure 1. The red, blue and green lines indicate
The permeate fraction is then centrifuged in a 50K approximate molecular weight ranges and represent the theoretical MWCOs.
device. This process is repeated for the 30K device. Colored dashed boxes represent size specific protein compartments.

54
The traditional methodology employing use of a References
single MWCO to remove HMW and enrich for
1. Forssmann WG, et al. J Chromatogr A
the LMW resulted in impure fractions compared
1997;776:125-132.
to those from serial enrichment.
2. Plebani M, et al. Pancreas 2002;24:8-14.
Conclusions 3. Sobrinho LG, et al. Clin Endocrinol (Oxf)
Protein fractionation using centrifugal ultrafiltration 2003;58:686-690.
devices is a simple and rapid means of reducing 4. Werner MJ. Chromatogr 1966;25(1):63-70.
sample complexity and compartmentalize proteins 5. Kent UM. Methods Mol Bio 1999;
based on molecular weight. Serial enrichment is 115:11-18.
faster than a direct single device enrichment and
protein fractions resulting from serial enrichment
have greater purity than those from a single device
method. Regenerated cellulose membranes out-
perform PES membranes in an enrichment paradigm.
Ultrafiltration is a potential alternative to gel filtration
chromatography for size based protein enrichment.

Figure 3. Serial enrichment is more efficient than Figure 4. Regenerated cellulose

direct enrichment in terms of purity and time membranes offer optimal separation
compared to PES
Time Required:

P ro t o c o l s f o r P ro t e in s P ro te i n E n rich m e n t U s i n g U l t r af i l t r a t io n
Serial Direct 100
100 min 130 min IgG (0.1 mg/mL)
Recovery of Cytochrome C (%)

90
(150300 L) (500600 L)
80 IgG (1.0 mg/mL)
M S R100 R50 R30 R10 M S R30 R10 70
60
50
100 kDa 40
30
20
10
0
Ultracel PES Device A PES Device B
50 kDa
30 kDa 30 kDa Regenerated cellulose membranes (Ultracel mem-
Light brane) outperform polyethersulfone (PES) membranes
Chains
used for enrichment. A binary mixture containing
Cytochrome c (1 mg/mL) and 155 kDa IgG (0.1 or
10 kDa 10 kDa
1.0 mg/mL) was centrifuged using 100K MWCO
devices. Cytochrome c recovery was measured in
M: Marker R50: 50 kDa Rententate
the permeates. Significantly greater Cytochrome c
S: 50% Adult Serum R30: 30 kDa Retentate
recovery was observed using Ultracel membrane
R100: 100 kDa Retentate R10: 10 kDa Permeate when the system was challenged with high levels
of IgG (1.0 mg/mL).
Direct enrichment is not recommended for unknown or highly concentrated
samples. The figure shows 50% adult bovine serum enriched directly
(right panel) using an Amicon Ultra-4 30K MWCO device. The device was
centrifuged for more than 2 hours at 1500 x g to achieve a final volume of
~500 L, an 8X concentration. Presence of BSA and other higher molecular
weight contaminating proteins clearly show the benefits of using a serial
enrichment strategy (left panel) as described in Figure 1. Permeates collected
from the 30K (R10) devices were concentrated with 10K devices prior to
protein visualization. An unknown protein ~12 kDa was purified using a
serial method, but not direct enrichment.

55
 Urine Concentration with
Amicon Ultra Centrifugal Filters
Introduction
The measurement of specific proteins in urine is
important for the diagnosis and management of
disease states. In most cases, the content of these
proteins in urine is too low to be detected and
needs to be concentrated. Amicon Ultra-4 devices
can be used to concentrate urine samples prior to
clinical laboratory analyses. For example, patients
with multiple myeloma exhibit a proliferation of one
antibody-producing plasma cell, which leads to
excess production of free immunoglobulin light
chains known as Bence-Jones proteins. After sample
enrichment in the Amicon Ultra-4 10K NMWL
device, immunofixation electrophoresis can be used
to identify free light chains (Bence-Jones proteins)
in urine by forming a light chain-antibody complex.
Also, agarose electrophoresis can be used to
quantitate light chains and identify additional low
molecular weight proteins such as albumin, a-1
globulins, transferrin and IgG that can be present
in renal tubular disorders. Ultrafiltration of urine
samples in Amicon Ultra-4 devices provides
reproducible, high sample recovery for electropho-
P ro t o c o l s f o r P ro t e in s Uri n e C o n ce n t r a t io n
retic analyses, usually in 45 minutes or less.
56
Materials
Amicon Ultra-4 device, 4 mL, 10K NMWL
Centrifuge with fixed-angle or swinging bucket
rotor capable of 3400 x g
Kit for microprotein determination
(i.e. Sigma cat. no. 610-A/Brilliant blue G/
Coomassie blue)
Pipetter with 200 L tip
Electrophoresis (agarose gel) and immunofixation
equipment with apparatus and reagents
Method
1. Determine the total protein in a 24-hour urine
specimen.
2. Fill Amicon Ultra-4 device with 4 mL of urine.
3. Centrifuge at 3400 x g for 3045 minutes
(approximately 2550 L concentrate volume).
This produces up to a 160-fold increase in
concentration.
4. Insert a pipetter into the bottom of the filter unit
and withdraw the concentrated sample.
5. Perform agarose electrophoresis on the concen-
trate to quantitate light chains and identify other
proteins. Determine the percentage of light
chains with respect to the total number of
components in the urine. Then multiply the
percentage of light chains by the total 24-hour
protein concentration (grams per 24-hour volume).
6. Perform immunofixation electrophoresis on the
concentrate to identify light chains.
Acknowledgements
Research using Amicon Ultra devices for urine
concentration in this protocol was conducted by
Mark Merchant, Ph.D. at Helena Laboratories,
Beaumont, TX.
References
1. Tietz, N. Clinical Guide to Laboratory Tests,
2nd ed. Philadelphia: Saunders, WB;
1990;362363.
2. Kahns L. Clinical Chemistry 1991;37:
15571558.
3. Cleveland Clinic homepage. Accessed July
2002. www.clevelandclinic.org/myeloma/
DiagnosisAndTreatmentOf MultipleMyeloma.html
4. Christenson RH, et al. Clinical Chemistry
1983;29(6):10281030.
5. Christenson RH, and Russell ME. Clinical
Chemistry 1985;31(6):973.
Additional Notes
1. Amicon Ultra devices can also be used to
concentrate serum, plasma and cerebrospinal
fluid for similar analyses. A concentration of
approximately 20 mg/mL is required in order to
detect free light chains from diseased patients by
agarose electrophoresis. Detection by immuno-
fixation electrophoresis is 10 times more sensitive
than by agarose electrophoresis.
2. Normal heterogeneous immunoglobulins may
also be seen in urine concentrate with immuno-
fixation electrophoresis. This ladder effect is
comprised of microheterogenous light chains.
Bence-Jones proteins may be within this ladder.
To verify the presence of Bence-Jones proteins
requires additional analysis by two-dimensional
electrophoresis.
3. If there is excess antigen, dilution of the concen-
trate will be required until equilibrium is achieved
between the antigen (Bence-Jones protein) and
the antibody.
4. Millipore also offers static concentrators
(Minicon devices) for concentration of
Bence-Jones protein in urine.
P ro t o c o l s f o r P ro t e in s Uri n e C o n ce n t r a t io n
57
 Use of Centrifugal Filter Devices as
an Alternative to Stirred Cells
Introduction
Stirred cell devices have been successfully used to
purify proteins from large volumes of solution for
many years. However, the need for assembly and
cleaning between uses, and the lengthy separation
times, can present difficulties for active, or under-
staffed, laboratories.
Millipores large volume centrifugal filter devices
P ro t o c o l s f o r P ro t e in s U s e of C e n t rif u g a l F i l te r D ev ice s a s a n A l te rn a t i ve to St i rre d C e l l s
Abstract
This study aims to achieve purification and concen-
tration of a fusion protein composed of the alpha
and gamma subunits of the human high affinity
IgE receptor. The alpha-gamma fusion protein is
purified and then coupled to Sepharose (GE)
beads to produce an affinity column for isolating
human IgE antibodies.

come preassembled and ready to use. Individual

devices are available for processing 20 or 70 mL
volumes. For larger volumes, multiple devices can
be spun simultaneously. Spin times are typically
measured in minutes. And unlike stirred cells,
centrifugal devices run unattended. There is no
need to refill a reservoir and, therefore, less risk
of adventitious contamination.
The following protocol demonstrates the use
of Centricon Plus-70 centrifugal filter devices to
purify and concentrate proteins from large volumes
of solution.
Figure 1.
Method
The alpha and gamma subunit components of the
high affinity IgE receptor were transfected into
mouse myeloma cell line NS0 and the secreted
fusion protein was purified on a Protein G column.
The eluted proteins were concentrated from
volumes of 5001000 mL to 510 mL using a
Centricon Plus-70 centrifugal filter device in a
swinging bucket rotor at 3000 x g for 10 minutes
(60 mL per filter unit). Buffer exchange was also
performed subsequently using the same units in
tissue culture grade sodium azide-free PBS. The
concentrated samples were then filter-sterilized
under sterile conditions using a 0.2 m pore filter.

140
Results
120
Spectrophotometric evaluation of alpha-gamma
Absorbance Units

100 fusion protein recovery using Centricon Plus-70

80 devices showed a result of 18.8 mg/L of superna-
60 tant. HPLC trace analysis revealed the chromato-
gram shown in Figure 1.
40

20
Acknowledgement
0
0 5 10 15 20 25 30
Protocol courtesy of GKT School of Biomedical
Time (min) Sciences, London.

HPLC profile of alpha-gamma fusion component of the human FcRI

58
Protocols for
Purification of DNA Sequencing Reactions . . . . 60

Concentrating and Desalting DNA or RNA . . . . 63

Nucleic Acids Preparing Samples for Forensics ID Analysis . . . 66

Concentration of Genomic DNA for

Forensic Analysis . . . . . . . . . . . . . . . . . . . . . . 67

Purification of PCR Products . . . . . . . . . . . . . . . 68

Quantitative Recoveries of Nanogram

Amounts of Nucleic Acids . . . . . . . . . . . . . . . . 70

RNA Purification and Preparation of

Fluorescent cDNA Probe from Human mRNA . . 72

Purification of In Vitro Synthesized mRNA . . . . . 76

Effect of Centrifugal Ultrafiltration on

Large Fragment DNA Integrity . . . . . . . . . . . . . 78
DNA Extraction from Agarose Gels . . . . . . . . . 80

PCR Purification . . . . . . . . . . . . . . . . . . . . . . . 82

Enzyme Removal . . . . . . . . . . . . . . . . . . . . . . 84

P ro t o c o l s f o r Nuc l e ic Ac id s

59
 Use of Microcon 100 Centrifugal Filter
Devices to Purify DNA Sequencing
Reactions
Introduction
DNA sequencing reactions performed using ABIs
BigDye terminator chemistries require the removal
of unincorporated dye-terminators and excess salt
prior to injection into a sequencer. The traditional
method for purifying DNA sequencing reactions is
ethanol precipitationa labor intensive method
requiring multiple spins, incubation, and drying that
can take up to an hour. Care must also be taken to
retain the resulting pellet.
Microcon 100 devices can remove contaminat-
ing salts and unincorporated dye terminators from
P ro t o c o l s f o r Nuc l e ic Ac id s P u rif ic a t io n of D N A S e q u e n ci n g Re a c t io n s
DNA sequencing reactions and deliver SEQ quality
and read lengths equal to or better than ethanol
precipitation in 10 minutes. As capillary-based
DNA sequencers become more prevalent, the need
for single sample purification of SEQ reactions also
increases.
The first protocol achieved a time savings of
50 minutes over the traditional ethanol precipitation
method while a second protocol, which relies on
less vigorous centrifugation, took approximately
as long as ethanol precipitation but excelled in
experiments in which it was desirable to read
close to the primer.
Equipment
Instrument: ABI sequencer cat. no.3700
Reagent: ABI BigDye Terminator v3.1
Injection parameters: existing parameters
developed for Montage Injection solution.
Data output: Graphs and electropherograms
that show A) removal of dye-terminators (blobs);
B) Read length (Phred 20 scores); C) Primer
60
Method
DNA was isolated according to standard protocols
and amplified for sequencing analysis on an ABI
model 3700 DNA sequencer using polymerase
reagents and ABIs BigDye Terminator v. 3.1.
Sample was purified using two experimental
protocols. Results were assessed using electrophero-
grams, Phred 20 read lengths, and estimation of
dye blobs.
Microcon 100 protocol
1. Sequence Product (10 L) + 100 L of Montage
injection solution in a Microcon 100 device
2. Spin for 2.5 minutes at 12,000 x g
3. Add 100 L of injection solution to device
4. Spin for 2.5 minutes at 12,000 x g
5. Resuspend in 20 L of injection solution then
incubate for 2 minutes
6. Invert spin for 2 minutes at 1,000 x g
7. Inject into sequence analyzer
The above steps were completed in 10 minutes
at least one third the time required for ethanol
precipitation.
Results
Key parameters for DNA sequence reactions
include quality, read length, sequencing accuracy,
ability to read close to the primer, and the presence
of dye blobs. The first three factors may be assessed
using Phred 20 scoring, which is performed auto-
matically via software. Phred 20 corresponds
roughly to a 99% probability that a base call is
accurate. Ability to read close to the primer is
measured by the Primer + value, which relies on
both software and visual verification. Dye blobs
are artifacts of unknown origin, occurring during
sequencing or cleanup, that may overshadow
peaks. Dye blob assessment is typically performed
by visually examining the electropherogram.
Table 1. Results from a sequence purification
using the Microcon 100 device
Dye Terminator Phred 20 Dye Blobs Primer +
1/2X 646 59 0.4 11 10
1/8X 656 42 None observed 87
n=48

Figure 1. Electropherogram of a 1/2X SEQ reaction, purified using the Microcon 100 protocol

P ro t o c o l s f o r Nuc l e ic Ac id s P u rif ic a t io n of D N A S e q u e n ci n g Re a c t io n s
Microcon 100 at 12,000 x g Phred 20684 Primer + = 8 Dye blobs = 0

61
 Conclusion Table 2. Results from a typical sequence purification
Microcon 100 ultrafiltration devices represent a using an alternate protocol
rapid and efficient alternative to ethanol precipita- Dye Terminator Phred 20 Dye Blobs Primer +
tion when preparing DNA for sequencing analysis. 1/2X 627 83 0.6 10
The high-spin protocol outlined above affords a 1/8X 653 73 None observed 1.7 3
significant reduction (10 minutes vs. 60 minutes) in Ethanol 605 88 0.5 2.6 4
sample preparation time. For experiments where low
Prime + is required, researchers can employ a low-
speed centrifugation protocol that provides read
lengths equal to or better than ethanol precipitation,
with far less opportunity for user error.

Figure 2. Electropherogram of a 1/8X SEQ reaction, purified using an alternate protocol

P ro t o c o l s f o r Nuc l e ic Ac id s P u rif ic a t io n of D N A S e q u e n ci n g Re a c t io n s

Microcon 100 at 500 x g Phred 20 = 622 Primer + = 1 Dye blobs = 0

62
Concentrating and Desalting
DNA or RNA with Microcon and
Amicon Ultra Centrifugal Filters
Introduction Millipore ultrafiltration membranes are character-
ized with well-defined, globular solutes (proteins).
Centrifugal filter devices serve as powerful tools
Typically, the nominal molecular weight limit
in molecular biology applications, such as DNA
(NMWL) for a membrane is the point at which over
or RNA concentration and desalting procedures.
90% of a solute with that molecular weight will be
Ultrafiltration (UF) is a pressure-driven, convective
retained. To process DNA or RNA, the membrane
process that uses semipermeable membranes to
needs to be characterized according to the number
separate species by molecular size and shape.
of nucleotides in the fragment. Polynucleotides
UF is highly efficient, allowing for concentration
(DNA and RNA) have tertiary structures that are
and desalting at the same time. Unlike the use of
ordinarily more extended than those of typical
chemical precipitation methodologies (i.e., ethanol,
globular proteins of similar size. Millipore has
phenol/chloroform), there is no phase change,
determined nucleotide cut-offs (based on the
which often denatures labile species. DNA and
number of bases or base pairs in a fragment of

P ro t o c o l s f o r Nuc l e ic Ac id s C o n ce n t r a t i n g a n d D e s a l t i n g D N A o r R N A
RNA samples with starting concentrations as low as
DNA or RNA) that correspond to the NMWL of
5 ng/mL can be routinely concentrated in minutes
each of their low binding membranes. The nucleo-
with 99% recovery of starting material, and without
tide cut-off (NCO) indicates the fragment length of
the use of co-precipitants. Centrifugal concentrator
single- or double-stranded DNA or RNA that one
devices are ideal for separating high and low-
would expect to recover at 90% efficiency with a
molecular weight species. Ultrafiltration can also
unit of the named NCO. It is best to choose the
be used to change solvents by diafiltration. In this
NCO with about half the length of the fragment
process, the sample is concentrated, then diluted
of interest. For example, selecting a 30K NMWL
to the original volume with the desired buffer and
membrane for a 50 base pair fragment generally
concentrated again, thus washing out the
results in 90% product recovery. A 10K NMWL
original solvent.
membrane would provide for closer to 100%
recoveries but would take much longer to process
the sample. For nucleic acid samples >500 base
Table 1. Nucleotide cut-off guidelines for pairs, a 100K NMWL membrane is appropriate.
Microcon centrifugal devices (based on Table 1 offers guidelines for DNA/RNA retention
>90% recovery of nucleic acids) based on the nucleotide content of single- and
Single-Stranded* Double-Stranded double-stranded pieces. For example, more than
Nucleotide Nucleotide 90% of a single-stranded 30-mer will typically be
NMWL Cut-off (bp) Cut-off (bp)
retained by a Microcon 10K NMWL.
3K 10 10 Concentrating dilute DNA solutions is a key step
10K 30 20 for many subsequent preparative and analytical
30K 60 50 procedures. For example, standard plasmid
50K 125 100 preparations involving cesium chloride, equilibrium
100K 300 125 centrifugation, and gel filtration yield DNA in large
*Single-stranded nucleic acids with extensive secondary volumes that require concentrating prior to precipita-
structure will be better retained than those without. tion. DNA concentration is also necessary in
purifying restriction fragments from gels.

63
 There are three major techniques currently
available for concentrating nucleic acids:
repeated extractions with n-butanol
adsorption to ion exchange resin,
followed by high salt elution
lyophilization
The first method has the disadvantage that
n-butanol concentrates all solutes, including salt,
which tends to co-precipitate with DNA upon
addition of ethanol. The second method, ion
exchange, aside from requiring buffers of various
ionic strengths, yields DNA in a high salt solution.
The third method, lyophilization, increases the
concentration of buffer components, which can
result in degradation of nucleic acids.
Ultrafiltration membranes retain DNA or RNA but
are permeable by smaller ionic buffer components.
Ultrafiltration alone does not change buffer compo-
sition. The salt concentration in a sample concen-
trated by Microcon centrifugal filter devices will be
the same as in the original sample. For desalting,
the concentrated sample is diluted with water or
buffer to its original volume and spun again in a
process called diafiltration. This removes the salt
by the concentration factor of the ultrafiltration.
For example, if a 500 L sample containing
100 mM salt is concentrated to 25 L (20X
concentration factor), 95% of the total salt in the
sample will be removed. The salt concentration in
the sample will remain at 100 mM. Rediluting the
sample to 500 L will bring the salt concentration
to 5 mM. Concentrating to 25 L once more will
remove 99% of the original total salt. The concen-
trated sample will now be in 5 mM salt. For more
complete salt removal, an additional redilution and
spinning cycle will remove 99.9% of the initial salt
content (see Table 2).

Methods
P ro t o c o l s f o r Nuc l e ic Ac id s C o n ce n t r a t i n g a n d D e s a l t i n g D N A o r R N A

Table 2. Desalting and recovery with Microcon Microcon Device

30K NMWL devices
1. Select a Microcon unit with nucleotide cut-off
Spin % DNA % CsCl equal to or smaller than the molecular size of the
Number Recovered Removed
nucleic acid you want to retain (refer to Table 1).
1 88 99.9
2. Insert Microcon sample reservoir into one
2 89 100
of the two vials provided for each unit.
3 88 100
3. To concentrate (without affecting salt concentra-
Using 1 mL of 25 g/mL E. coli DNA in 6 M CsCl, sample was repeatedly con- tion): Pipette up to 500 L of DNA or RNA
centrated to 0.1 mL. Sample spun at 2000 x g in a 45 degree fixed angle rotor.
The concentrated sample was reconstituted to original 1 mL volume by adding
sample into the reservoir. Spin for recommended
50 mm Tris. After three spins, CsCl concentration was reduced by four orders of time, not exceeding recommended g-force
magnitude. guidelines shown in Table 3.
4. To exchange salt: Add the proper amount of
appropriate the diluent to bring the concentrated
Table 3. Recommended g-force and spin time sample to 500 L. Spin for the recommended
for Microcon devices time, not exceeding g-force shown in Table 3.
To achieve lower salt concentration, repeat the
Maximum Spin Time (min) Spin Time (min)
NMWL G-Force Rating at 4 C at 25 C entire step as necessary. NOTE: Do not let filtrate
3K 14,000 185 95 vial overfill.

10K 14,000 50 35 5. Remove reservoir from vial and invert into a

30K 14,000 15 8 new vial (save filtrate until sample has been
analyzed).
50K 14,000 10 6
100K 500 25 15 6. Spin for 2 minutes at 5001000 x g to recover
nucleic acid in the vial.
7. Remove reservoir. Cap vial to store.

64
Amicon Ultra Device Table 4. Nucleotide cut-off guidelines for Amicon Ultra-4
1. Select an Amicon Ultra unit with a nucleotide A. Nucleotide cut-off guidelines for single stranded DNA
cut-off equal to or smaller than the molecular size
Single-Stranded NMWL
of the nucleic acid you want to retain (refer to
Nucleotide Cut-off (bp) 3K (40 min) 10K (20 min)
Table 4).
10 98 97
2. To concentrate (without affecting salt concentra-
15 96 95
tion): Pipette up to 4 mL of DNA or RNA sample
20 94 94
into the reservoir. Spin for the recommended time,
25 96 96
not exceeding g-force shown in Table 5.
3. To exchange salt: Add the proper amount of the B. Nucleotide cut-off guidelines for double stranded DNA
appropriate diluent to bring the concentrated NMWL
Double-Stranded
sample to 4 mL. Spin for the recommended time, Nucleotide Cut-off (bp) 30K 50K 100K
not exceeding g-force shown in the Table 5. 137 95 95
To achieve lower salt concentration, repeat the
301 94
entire step as necessary. NOTE: Do not let filtrate
657 99
vial overfill.
1159 100 97
Based on >90% retention. Spinning 50 and 100K devices at higher than
recommended g-force may result in lower recovery of DNA.

P ro t o c o l s f o r Nuc l e ic Ac id s C o n ce n t r a t i n g a n d D e s a l t i n g D N A o r R N A
Table 5. Recommended g-force and spin time
for Amicon Ultra devices
Maximum Spin Time (min)
NMWL G-Force Rating at 25 C
3K 7,500 40
10K 7,500 20
30K 5,000 10
50K 2,000 20
100K 2,000 10

65
 Preparing Samples for Forensics
Identification Analysis Using
Microcon Centrifugal Devices
Centrifugal filter devices with ultrafiltration (UF)
membranes are used by forensics laboratories to
isolate genomic DNA for human identification. The
source materials are typically blood stains and/or
other bodily fluids obtained from crime scenes,
criminal suspects, or human remains.
The isolated genomic DNA is used to identify
individual persons based on their patterns of Short
P ro t o c o l s f o r Nuc l e ic Ac id s P re p a ri n g S a m p l e s f o r F o re n s ic s I d e n t if ic a t io n A n a l y s i s
Several suppliers offer STR assay kits. Each
supplier offers several versions of their kits with
different protocols, modes of operation, and
detection systems to suit the users needs. Millipores
UF-based centrifugal devices are specified
in several of these protocols (AmpFLSTR Profiler,
Applied Biosystems; GenePrint STR and GenePrint
Fluorescent STR Systems, Promega).

Tandem Repeats. STRs are polymorphic DNA loci

that contain repeated DNA sequences. Because the Use of UF-based Centrifugal Devices in
repeats can vary from two to seven bases in length, New York City
many different alleles are possible for each locus. Depending on the type of evidence under investiga-
The parallel analysis of 9 to 13 polymorphic tion, the Department of Forensic Biology in New
STR loci can unequivocally identify an individual. York City had been preparing genomic DNA by
either (1) organic extraction (phenol/chloroform/
isoamylalcohol) followed by alcohol precipitation
Figure 1. or (2) direct lysis in the presence of Chelex beads
Genotype analysis of a (Bio-Rad) with no further purification.
semen sample before The Department of Forensic Biologys laboratory
A
and after concentration has replaced the ethanol precipitation step with
by a Microcon centrifu-
gal filter. Electrophero-
Microcon centrifugal filters with a 100K NMWL
gram A shows a partial ultrafiltration membrane. The centrifugal devices
profile with a high provide a faster way to concentrate the purified
molecular weight loci
DNA without the use of toxic chemicals.
in each color missing.
Electropherogram B The laboratory also uses the Microcon 100K
shows a full profile and NMWL device after Chelex extraction to remove
higher peak heights. inhibitors that can adversely affect PCR amplifica-
Amounts amplified were
tion. In addition, Chelex extraction of small blood-
0.62 ng for electrophe-
rogram A and 1 ng for stain samples often results in DNA concentrations
electropherogram B. below detection limits. Concentrating extracted
B The sample was extract- samples with Microcon 100K NMWL devices
ed with Chelex beads.
The peaks are labeled
improves the success rate of STR amplification and
with allele name, size yields a full STR allele profile even with minute
in base pairs, and blood or semen stains. According to the New York
fluorescent peak height.
City Department of Forensic Biology, several of the
The improvement in
results is due not only peaks shown in Figure 1 would have been below
to the increased DNA the detection threshold and identification would
input but also to the have been negative or inconclusive without sample
concentration step
cleanup using Microcon devices.
following extraction.

66
Concentration of Genomic DNA
for Forensic Analysis Using
Amicon Ultra-4 50K Centrifugal Devices
Introduction Protocol Recommendations for
Millipores Amicon Ultra-4 centrifugal filter devices Genomic DNA Isolation
provide fast ultrafiltration, with the capability for Dilute sample to 2.0 mL with TE Buffer
high concentration factors. The device design (10 mM Tris, pH 7.6, 1 mM EDTA)
incorporates the Millipore Ultracel low binding
Centrifuge at 2,000 x g for 20 minutes
regenerated cellulose membrane, providing
at room temperature
excellent sample recoveries from dilute and complex
sample matrices. The Amicon Ultra device is Repeat for the number of washes desired
configured in a vertical design, which minimizes Note: The centrifugation time is based on samples

P ro t o c o l s f o r Nuc l e ic Ac id s C o n ce n t r a t io n of G e n o m ic D N A f o r F o re n s ic A n a l y s i s
solute polarization and subsequent fouling of the containing up to 2 g of genomic DNA. For forensics
membrane. The vertical design and available samples, users will likely need to determine the
surface area provide for fast sample processing, appropriate centrifugation time as many factors
high retentate recovery (typically greater than (e.g., DNA concentration and temperature) will
90% of dilute protein or DNA concentrate), and affect centrifugation time.
the capability for very high concentration factors
(>80-fold). The Amicon Ultra-4 devices are suitable
for use with dilute nucleic acid samples which can
be quickly ultrafiltered, allowing for fast separation
Amicon Ultra has enabled our testing lab
of genomic DNA from low molecular weight (MW)
to process samples quicker and with a
compounds.
higher convenience and confidence as the
The Amicon Ultra device design is compatible
decreased number of handling steps allows
with the multiple spin recovery assays used for the
us to be free of cross-contamination.
purification of genomic DNA for forensic analysis.
The concentrate is collected from the filter unit Professor Miguel Paredes,
sample reservoir using a pipetter, while the ultrafil- Barcelona Laboratory of Instituto Nacional
trate can be collected in the provided centrifuge de Toxicologia y Ciencias Forenses
tube if desired. The filter device can be spun in a
swinging bucket or a fixed angle rotor. The Amicon
Ultra devices come fully assembled in a centrifuge
tube, ready for sample use.

67
 Purification of PCR Products
with an Amicon Ultra Device
Introduction
To enable the use of DNA fragments generated by
the Polymerase Chain Reaction (PCR) in downstream
applications, unincorporated primers, nucleotides
and contaminating salts must to be quantitatively
removed. The application of ultrafiltration to PCR
clean-up is well established and represents a
convenient and efficient approach to generate
concentrated, purified PCR products suitable for such
downstream applications such as cloning or DNA
sequencing. Millipore offers a number of products
designed for the purification of PCR products in
96-well and 384-well plate and 1-up formats.
The protocol and data shown here demonstrate the
utility of Amicon Ultra-4 devices for the purification
of PCR products. By extension, these data can be
used to select an Amicon Ultra with the appropriate
molecular weight cut-off (MWCO) for use in the
purification of DNA used in other applications.
P ro t o c o l s f o r Nuc l e ic Ac id s P u rif ic a t io n of P C R P ro d u c t s
Considerations
For applications involving nucleic acids, strand
length is the most useful parameter for selecting the
Amicon Ultra device appropriate for a particular
application. However other parameters including,
DNA concentration, the magnitude of the driving
force (g-force) and the salt concentration all act in
concert to affect DNA recovery. When purifying
PCR reactions for example, optimal yield can be
Figure 1. PCR recovery with
Amicon Ultra-4 50K device
100
Recovery (% of Challenge)
80
60
40
20
0
(n=8) 137 301 657
PCR Fragmentation Size (bp)
68
Figure 2. Effect of MWCO and g-force
on the recovery of a 137 bp PCR product
using Amicon Ultra-4 100K device
100
% PCR Recovery
80
60
40
20
0
2,000 x g 5,000 x g 7,500 x g
achieved via dilution of the starting material
and running at a relatively low g-force. For
PCR fragments, an Amicon Ultra-4 device with
a 50K MWCO membrane generally strikes the
best balance between speed and recovery. If the
DNA sample is in a buffer containing high salt and
cannot be diluted, or the device is run at a higher
than recommended g-force, a significantly reduced
DNA recovery will likely be observed. Under these
conditions, higher DNA or RNA recovery can
be achieved by using the Amicon Ultra with the
next tighter membrane, albeit with a much longer
processing time. Note that, for maximal removal of
unincorporated single-stranded primers from double-
stranded DNA fragments, the molecular weights of
the primer and DNA fragment should differ by at
least an order of magnitude.
Method
DNA fragments (137, 301, 657 and 1159 bp)
were generated by PCR using plasmid DNA as
the template. After pooling to minimize device-
to-device variability, 100 L aliquots of the PCR
reactions were diluted with 2.0 mL of TE Buffer
(10 mM Tris-HCL, pH 8.0, 1 mM EDTA) and
added to an Amicon Ultra-4 device with a 50K
MWCO membrane. The samples were centrifuged
1159 at 2,000 x g for 20 minutes, an additional 2.0 mL
of TE buffer was added to the sampled and the PCR
products were concentrated by a second 20-minute
spin at 2,000 x g. The purified PCR products were
collected by pipette and the recovered DNA
quantified by a fluorescent SYBR Green I assay
(Molecular Probes). The final sample volume was
87 15 L. The recoveries shown in Figure 1
demonstrate the high recovery that can be obtained
from PCR reactions using this approach.
The recovery of PCR fragments purified by
ultrafiltration can be challenging when the resulting
DNA fragments are <1000 bp in length. The
amount of DNA recovered after purification can
be maximized by using an Amicon Ultra device
with a lower MWCO membrane (e.g., 50K versus
100K MWCO) and by centrifuging the devices at
2,000 x g as shown in Figure 2 for a 137 bp PCR
fragment. Although increasing the g-force will speed
processing time, it will do so at the expense of DNA
recovery. For larger DNAs, the effect is less
dramatic (data not shown).
The quantitative removal of single-stranded DNA
primers or oligonucleotides from PCR reactions is
critical for downstream applications. In order to
demonstrate the ability of Amicon Ultra devices to
efficiently remove primers, unpurified PCR products
were spiked with 20 pmoles of a DNA reporter
primer that had been labeled with fluorescein on its
5 end. This primer (TCAG)5 was designed such that
it had minimal secondary structure. After purification
of the PCR products with an Amicon Ultra-4 device,
the amount of primer remaining in the sample was
determined by measuring fluorescence against a
standard curve. The average primer removal in this
experiment was 97.7 1.4%. This level of primer
removal has been shown to be sufficient for DNA
sequencing and other important PCR-based
applications.
Figure 3. Amicon Ultra device efficiently
removes unincorporated PCR primers
100
Removal (% of Challenge)
80
60
40
20
0
137 301
PCR Fragmentation Size (bp)
Ultrafiltration-based purification relies on size
exclusion to affect the separation of single stranded
primers from the double stranded PCR fragments.
As discussed above, the major challenge to
optimizing ultrafiltration for PCR purification is
with small, <1000 bp PCR products. Similarly, the
efficiency of primer removal becomes more
challenging as the size of the single stranded primer
increases. As shown in Figure 4, the ability of an
Amicon Ultra device with 30K MWCO membrane
to remove primers is significantly diminished as the
primer length approaches 50 bases. In contrast,
when a 50K MWCO membrane is used, primer
removal remains high and is consistent across the
range of PCR primers evaluated. Whereas the use
of a 100K would be expected to quantitatively
remove all but the largest of primers, purification
of small (e.g., <300 bp) PCR products is suboptimal
when using a 100k MWCO membrane. Therefore,
an Amicon Ultra-4 device with a 50K MWCO
membrane provides the best balance of primer
removal and recovery when used for PCR
purification.
Conclusions
P ro t o c o l s f o r Nuc l e ic Ac id s P u rif ic a t io n of P C R P ro d u c t s
The data presented herein demonstrate the utility of
Amicon Ultra-4 devices for the purification of PCR
products. By using devices with a 50K MWCO,
users can expect to routinely obtain both high
recoveries and efficient primer removal that will
facilitate downstream applications. Although the
analysis performed for this study focused on PCR
purification, the general principles are applicable
to purification of other nucleic acid preparations.
Figure 4. Effect of MWCO on primer
removal in Amicon Ultra devices
100
Removal (% of Challenge)
80
60
40
20
0
20 25 48
Primer Length (Bases)
657 1159 Amicon Ultra, 50k Amicon Ultra, 30k
69
 Quantitative Recoveries of Nanogram
Amounts of Nucleic Acids with
Microcon Centrifugal Filters
Introduction were then centrifuged at 12,000 x g in a fixed-
angle microcentrifuge at 4 C for 15 minutes. The
Molecular cloning experiments often require
supernatants were carefully removed and pellets
concentration of nanogram quantities of DNA.
resuspended in 50 L of TE buffer. The radioactivity
When dealing with such small amounts of nucleic
in the precipitates and supernatants was then
acids, the use of coprecipitants (tRNA or purified
determined by counting Cherenkov radiation. The
glycogen) is essential for effective DNA recovery1.
P ro t o c o l s f o r Nuc l e ic Ac id s Q u a n t i t a t i ve Re cove rie s of N a n o g r a m A m o u n t s of Nu cl e i c A ci d s

percentage of recovered DNA was calculated from

However, when carrier tRNA cannot be used (for
example, when DNA is to be labeled with 32P dATP
and polynucleotide kinase), the only method of
recovering DNA as a precipitate in ethanol has
been the ultracentrifugation method developed by
Shapiro2. Ultrafiltration, which has been traditionally
used to concentrate and desalt protein samples
simultaneously, can be an efficient alternative to
ethanol precipitation.
In this experiment, three popular ethanol
precipitation protocols for DNA and oligonucle-
otides are compared with ultrafiltration in Microcon
centrifugal filter devices. For a detailed discussion
on the effects of incubation time, temperature and
centrifugation parameters on ethanol precipitation,
see Reference 3.
Methods
Plasmid pBR322 was digested with EcoR I and the
3 recessed termini were filled in with alpha 32P
dATP, using Klenow fragment of DNA polymerase I.
A 25-nucleotide mixed base oligomer was radio
labeled by phosphorylation with bacteriophage T4
polynucleotide kinase.
Ethanol Precipitation
All DNA precipitations were performed in 250 L
volume. Each tube contained a given amount of
DNA, supplemented with 10 ng of radioactively
labeled pBR322 in 0.3 M sodium acetate buffer.
To precipitate the DNA, 2.5 volumes of 95% EtOH
were added to each tube. The content was well
mixed and incubated for the specified period of
time at 20 C or 70 C (dry ice). The solutions
precipitates and the initial counts. Each data point
in the tables represents the average of at least
three samples.
To precipitate the oligomer, 60 L of TE buffer
containing a given amount of 25-mer and 5 ng of
radiolabeled tracer was supplemented with 240 L
of 5 M ammonium acetate and 750 L of EtOH.
Subsequent steps were identical to those described
for DNA.
Ultrafiltration
A solution of DNA (500 L) was placed into a
Microcon 30K NMWL unit and spun at 12,000 x g
for 10 minutes. A 500 L solution of oligonucle-
otides in TE buffer was placed into a Microcon 3K
NMWL unit and spun for 45 minutes at 12,000 x g.
The retentates were recovered by inverting the units
and centrifuging at 5001000 x g for 2 minutes.
While concentrating the oligomer samples, it is
important to avoid high salt concentrations which
promote binding of single-stranded nucleic acids
to the cellulose-based ultrafiltration membrane.
The radioactivity in the retentates and filtrates
was determined by counting Cherenkov radiation.
Data represent averages of six samples each.
Results
Standard methods for ethanol precipitation of
nucleic acids and ultrafiltration are compared in
Tables 1 and 2. Recovery of DNA was assessed at
close to 100%, indicating that no significant amount
of nucleic acid was lost to the membrane or device
due to adsorption.

70
 In contrast, DNA recovery using EtOH precipita- References
tion varied from as little as 14% as in the case of
1. Wallace DM. Precipitation of Nucleic Acids.
DNA (10 ng/mL) precipitated for 15 minutes at
Methods in Enzymology 1987;152:418.
70 C to a maximum of 76% after overnight
incubation at 20 C. 2. Shapiro DJ. Quantitative Ethanol Precipitation of
Nanogram Quantities of DNA and RNA. Anal
Discussion Biochem 1981;110:22931.
Poor recovery of nucleic acids at very low concen- 3. Crouse J, Amorese D. Ethanol Precipitation:
tration with ethanol precipitation may be partially Ammonium Acetate as an Alternative to Sodium
due to the fact that small amounts of DNA do not Acetate. Focus 1987;9(2):35.
adhere well to the tube walls following sedimenta-
tion unless high g forces (ultracentrifugation) are
employed. The yield of DNA incubated at 70 C
is slightly reduced, in agreement with previous

P ro t o c o l s f o r Nuc l e ic Ac id s Q u a n t i t a t i ve Re cove rie s of N a n o g r a m A m o u n t s of Nu cl e i c A ci d s

studies. Also an overnight precipitation at 20 C
can significantly improve recovery. The ultrafiltration
devices were found to concentrate and desalt
nucleic acids effectively in one step resulting in
high recoveries and providing a quick, alternative
to ethanol precipitation.

Table 1. Efficiency of different methods for concentration of DNA

DNA Concentration
10 ng/mL 25 ng/mL 50 ng/mL 250 ng/mL 1000 ng/mL
Method % Recovery % Recovery % Recovery % Recovery % Recovery
EtOH, 70 C, 15 min 14 15 23 52 55
EtOH, 20 C, 30 min 13 20 25 60 72
EtOH, 20 C, 18 hrs 31 45 45 76 67
Microcon 30K NMWL Device 95 95 98 98 99

Table 2. Efficiency of different methods for concentration of oligonucleotides

Oligomer Concentration
10 ng/mL 25 ng/mL 50 ng/mL 250 ng/mL 1000 ng/mL
Method % Recovery % Recovery % Recovery % Recovery % Recovery
EtOH, 70 C, 15 min 4 4 5 6 33
EtOH, 20 C, 30 min 6 5 6 4 33
EtOH, 20 C, 18 hrs 17 20 15 30 58
Microcon 30K NMWL Device 93 94 93 95 95

71
 RNA Purification and Preparation of
Fluorescent cDNA Probe from Human
mRNA with Microcon Centrifugal Filters
Introduction
P ro t o c o l s f o r Nuc l e ic Ac id s R N A P u rif ic a t io n a n d P re p a r a t io n of F lu o re s ce n t cD N A P ro b e f ro m Hu m a n m R N A
When working with RNA, introduction of RNAse
contamination during sample preparation is of major
concern. Sample yield and integrity can impact the
efficiency of subsequent translations, hybridizations,
protein binding, antisense, or ribozyme studies. A
series of experiments was performed to determine
the effectiveness of using Amicon centrifugal
ultrafiltration devices from Millipore to concentrate
and diafilter RNA samples. The results indicate
remarkably high RNA recoveries and low adsorption
losses, especially with prior membrane passivation.
Microarrays are efficient tools that enable the
high throughput identification of genes that are
differentially regulated in response to disease, drugs
or other stimuli. With the completion of several key
genome sequencing projects, scientists now have
the ability to custom design DNA microarrays
specific to their research interests. The recent
advances in robotics, bioinformatics and detection
technologies have greatly simplified the manufacture
and analysis of microarrays.
However, the successful application of microarray
technology requires highly purified, fluorescently
labeled cDNA probes. The application of ultrafiltra-
tion technology to this challenge has resulted in
a robust, efficient and rapid method for the genera-
Figure 1.
Concentration of RNA transcript. RNA is intact
and recovered with high efficiency.
Lane 1: Starting material 1.4 kb
Lane 2: RNA concentrated in a Microcon
100K NMWL device
Lane 3: RNA concentrated in a
Microcon 30K NMWL device 0.68 kb
72
tion of high quality fluorescently labeled probes
suitable for use with microarrays. The protocol
below describes a method for the generation and
purification of fluorescently labeled probes using a
Microcon 30K NMWL centrifugal filter device.
RNA Sample Integrity
The primary objective of the study was to determine
the effect of centrifugation on RNA integrity. The
second objective was to determine any gross
changes in the RNA that could be a result of
contact of the sample with the ultrafiltration devices
themselves. An RNA transcript was made, using
MEGAscript T7 RNA polymerase (Ambion) and
DNA template Riboprobe Gemini (Promega)
according to manufacturers protocols. This transcript
served as the starting RNA sample for the experi-
ments discussed below. Figure 1 is an autoradio-
gram of labeled RNA. It compares an untreated
sample to samples that were concentrated in
Microcon 100K NMWL and 30K NMWL devices,
used directly as supplied. The Microcon 100K
NMWL unit was spun at a force of 3,000 x g and
the Microcon 30K NMWL unit at 12,000 x g. As
is evident from the autoradiogram, both unfiltered
samples are virtually indistinguishable from the
starting material.
1 2 3
Method
Generation and Purification of Fluorescently
Labeled Probes
1. To anneal primer, mix 2 g of mRNA or
50100 g total RNA with 4 g of a regular
or anchored oligo-dT primer in a total volume
of 15.4 L as shown in Table 1.
7. Neutralize by addition of 15 L of 0.1 N HCl.
8. Add 380 L of TE (10 mM Tris, 1 mM EDTA) to
a Microcon 30K NMWL device column. Next
add the 60 L of Cy5 probe and the 60 L of
Cy3 probe to the same Microcon device.
NOTE: If re-purification of Cy dye flow-through is
desired, do not combine probes until Wash 2.

P ro t o c o l s f o r Nuc l e ic Ac id s R N A P u rif ic a t io n a n d P re p a r a t io n of F lu o re s ce n t cD N A P ro b e f ro m Hu m a n m R N A
9. Wash 1: Spin column for 7 to 8 minutes at
2. Heat to 65 C for 10 minutes and cool on ice.
14,000 x g.
3. Add 14.6 L of reaction mixture each to Cy3
10. Wash 2: Remove flow-through and add
and Cy5 reactions as shown in Table 2.
450 L TE and spin for 78 minutes at
4. Incubate at 42 C for 1 hour.
14,000 x g. It is a good idea to save the flow-
5. Add 1 L SSII (RT booster) to each sample. through for each set of reactions in a separate
Incubate for an additional 0.5 1 hours. microcentrifuge tube.
6. Degrade RNA and stop reaction by addition
15 L of 0.1 N NaOH, 2 mM EDTA and
incubate at 6570 C for 10 minutes. If
starting with total RNA, degrade for 30 minutes
instead of 10 minutes.

Table 1. Preparation of primer

Primer Cy3 (L) Cy5 (L) Notes
mRNA (1 g/L) x y 2 g of each if mRNA; 50100 g if total RNA
Oligo-dT (4 g/L) 1 1 Anchored: 5-T T T T T T T T T T T T T T T T T T T T V N-3
Purified H2O (DEPC) to 15.4 to 15.4
Total volume 15.4 15.4

Table 2. Preparation of probe

Reaction Mixture Volume (L) Unlabeled dNTPs Volume (L) Final Concentration (mM)
5X first-strand buffer* 6.0 dATP (100 mM) 25 25
0.1 M DTT 3.0 dCTP (100 mM) 25 25
Unlabeled dNTPs 0.6 dGTP (100 mM) 25 25
Cy3 or Cy5
3.0 dTTP (100 mM) 10 10
(1 mM, GE Healthcare)
Superscript II 2.0 Purified H2O 15
(200 U/L, Gibco-BRL)
Total volume 14.6 Total volume 100
*5X first-strand buffer: 250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2

73
 11. Wash 3: Remove flow-through and add
450 L 1X TE, 20 g of Cot1 human DNA
(20 g/L, Gibco-BRL), 20 g polyA RNA
(10 g/L, Sigma cat. no. P9403) and
20 g tRNA (10 g/L, Gibco-BRL cat.
no. 15401-011). Spin 710 minutes at
14,000 x g. Look for concentration of the probe
in the Microcon device. The probe usually has
P ro t o c o l s f o r Nuc l e ic Ac id s R N A P u rif ic a t io n a n d P re p a r a t io n of F lu o re s ce n t cD N A P ro b e f ro m Hu m a n m R N A
a purple color at this point. Concentrate to a
volume of less than or equal to the volume
listed in the Probe and TE column in Table 3.
These low volumes are attained after the center
of the membrane is dry and the probe forms
a ring of liquid at the edges of the membrane.
Do not dry the membrane completely.
12. Invert the Microcon device into a clean tube
and spin briefly at 14,000 RPM to recover
the probe.
13. Select the appropriate row from Table 3. Adjust
the probe volume to the value indicated in the
Probe and TE column.
14. For final probe preparation add 4.25 L 20X
SSC and 0.75 L 10% SDS. When adding
the SDS, be sure to wipe the pipette tip with
clean, gloved fingers to remove excess SDS.
Avoid introducing bubbles and never vortex
after adding SDS. The probe is ready for
hybridization.
Table 3. Final probe preparation
Cover Slip Size Total Hybrid Volume (L) Probe and TE (L)
22 x 22 15 12
22 x 40 25 20
22 x 60 35 28
*20X SSC: 3.0 M NaCl, 300 mM NaCitrate (pH 7.0)
74
Results
RNA recovery is a function of the initial RNA
concentration and the buffer salt concentration.
Figure 2 shows RNA transcript recovery in
Microcon 100K.
NMWL unit as a function of RNA concentration.
For all concentrations evaluated, retentate recovery
is above 85%, even at an initial concentration as
low as 25 ng/mL.
Figure 2 also displays the material monitored in
the filtrate and on the membranes of the Microcon
units. (Membranes were removed and counted
without further treatment.) The amount of RNA in the
filtrate does not vary significantly as a function of
concentration. The RNA recovered on the mem-
branes varies from <1% at high initial concentrations
to 4% at the lowest concentration tested. Millipores
Amicon centrifugal ultrafiltration devices contain
low-binding cellulosic Ultracel-YM membranes.
Nitrocellulose is commonly used to immobilize RNA,
generally in high salt concentrations.
Figure 3 shows RNA recovery as a function of
buffer salt concentration. The data show that both
retentate and total recoveries fall as salt concentra-
tion increases. An increase in the amount of material
on the membrane is also seen (from 2% to 6%) as
the salt concentration increases from 10 to 500 mM.
Similar trends are seen in the use of Microcon
20X SSC* (L) 10% SDS (L)
2.55 0.45
4.25 0.75
5.95 1.05
100K NMWL devices, except that retentate
recoveries fall to 60% at 500 mM salt. It was Figure 2.
possible to count the devices themselves in addition 100%

to the filtrate and membrane. The results show a

80%
slight increase in counts on the membrane with

RNA Recovered
increasing salt concentration. Significantly more
60%
(from 9% to 17%) material remained on the device
as salt concentration increased. Passivation in- 40%

P ro t o c o l s f o r Nuc l e ic Ac id s R N A P u rif ic a t io n a n d P re p a r a t io n of F lu o re s ce n t cD N A P ro b e f ro m Hu m a n m R N A
creased RNA recovery to 80%, regardless of initial
20%
salt concentration. This is attributed to a decrease
in the amount of RNA adhering to the device. 0%
When the devices were passivated, RNA loss 0.025 0.25 0.5 1.0 5.0 10.0
RNA Concentration (g/mL)
due to adsorption was reduced to 2%, regardless
of salt concentration. Microcon Microcon Microcon
Filtrate Membrane Retentate

Conclusion RNA recovery in Microcon concentrators. RNA was concentrated as

The results shown here demonstrate that RNA described in text. Cherenkov counts of Microcon retentate, filtrate and
samples can be concentrated or diafiltered with membrane (n=3 units) were compared to total starting counts.

Microcon concentrators without loss of RNA

integrity. RNA recovery in the retentate is typically
>85%. As the salt concentration of the buffer system
Figure 3.
increases, RNA retentate recovery decreases,
principally due to RNA adhering to the device 100%

rather than to the membrane. RNA recovery from

80%
solutions with high initial salt concentrations can be
RNA Recovered

significantly improved by pre-treating the devices 60%

with a 5% SDS solution.
40%

Acknowledgements
20%
Fluorescent probe preparation protocol courtesy
of Patrick Brown, Max Diehn, and Ash Alizadeh, 0%
10 100 200 500
Stanford University School of Medicine. NaCl Concentration (mM)

Filtrate Membrane Device Retentate

Improvement in RNA recoveries using Microcon devices (n=6) which were

pre-treated with a 5% SDS solution.

75
 Purification of In Vitro Synthesized
mRNA with Microcon Centrifugal Filters
Introduction
In vitro transcription reactions employing T3, T7 or
SP6 phage-encoded RNA polymerases are widely
used to synthesize RNA from recombinant vectors
containing appropriate promoters. Production of
large amounts of specific RNA is valuable in the
preparation of hybridization probes and in vitro
translation studies; in the synthesis of ribozymes,
rRNA, SRP, antisense RNA and substrates for RNA
splicing; and in RNA-protein interaction studies.
Microcon centrifugal filters are well suited for
the purification of radiolabeled RNA transcripts1.
Ultrafiltration can simultaneously and efficiently
remove unincorporated ribonucleotides and salts
P ro t o c o l s f o r Nuc l e ic Ac id s P u rif ic a t io n of I n Vi t ro Sy n t h e s i ze d mR N A
from the transcripts and concentrate the RNA. RNA
molecules retain their integrity and are recovered
with high yields.
Purity of a transcript is especially important when
it is used in in vitro translation systems. Trace
amounts of ethanol, phenol, salts or excess cap
analog used during the synthesis of capped mRNA
can cause a dramatic decrease in translation
efficiency.
After the transcription reaction is complete,
template DNA is usually degraded by the addition
of DNase I. The RNA is purified by two phenol/
chloroform extractions followed by ethanol
precipitation. Other, less popular methods are gel
purification (used predominantly when separation of
full-length transcript from shorter RNAs is important,
e.g., ribonuclease protection assays) or LiCl
precipitation.
A series of experiments was performed in our
laboratory to determine the effectiveness of using
Microcon devices to purify in vitro synthesized
mRNA and in vitro translation studies. Results
indicate that ultrafiltration can efficiently remove
inhibitory contaminants from mRNA preparations,
leading to increased translational efficiencies.
76
Methods
RNA Transcription
For our studies we chose plasmid pGEM-luc
containing the luciferase gene (luc) in the center of
a multiple cloning cassette of the pGEM-11Zf (-)
plasmid (Promega). DNA template was linearized
with XhoI, followed by enzyme and salt removal by
diafiltration in Microcon 100K NMWL devices.
Linearized template was transcribed, using
MEGAscript kit (Ambion) according to the recom-
mended protocol. After the reaction was completed
(3 to 4 hours), template DNA was degraded with
DNase I and the reaction mix added to a Microcon
30K NMWL device filled with 450 L of water. The
device was spun for 20 minutes at 12,000 x g in a
temperature-controlled centrifuge at 4 C. Purified,
concentrated RNA was recovered by inverted spin.
For the preparation of capped transcript, cap
analog m7G (5) ppp (5) G (New England
Biolabs, Inc.) was included in the transcription
reaction and the level of GTP reduced (4:1 ratio
of cap analog to GTP). To purify the transcript by
phenol/chloroform extraction, the reaction mix
was diluted with water and a one-tenth volume of
ammonium acetate stop solution was added. The
mixture was extracted once with phenol/chloro-
form, followed by chloroform extraction. RNA was
precipitated with isopropanol and the pellet
resuspended in distilled water. Alternatively, LiCl
precipitation solution (one-half volume) was added
to the reaction mix, followed by incubation at minus
20 C for 1 hour. RNA was pelleted by centrifuga-
tion and dissolved in water. Size and integrity of
the in vitro transcription products were assessed by
running an aliquot of the purified RNA transcript on
a formaldehyde/formamide agarose gel. Ethidium
bromide was added to the RNA before lading on
the gel to stain the RNA sample and keep back-
ground fluorescence low2.
Translation In Vitro
In vitro translations were performed in the Flexi
Rabbit Reticulate Lysate System (Promega) according
to standard luciferase RNA translation conditions
with minor modifications (Rnasin Ribonuclease
inhibitor was omitted and 35S-methionine added).
Results of translation were analyzed by determina-
tion of percent incorporation of 35S-methionine and
fold stimulation, compared to controls without RNA.
Minimum acceptable stimulation was 8-fold.
Results
Aliquots of RNA transcript purified by different
methods (ultrafiltration, phenol extraction and LiCl
precipitation) were run on a denaturing agarose/
formaldehyde gel. Results are shown in Figure 1.
References
1. Krowczynska AM. BioSolutions 1993;2(1):12.
2. Ogretmen B, Ratajczak H, Kats A, Stark BC.
Biotechniques 1993;14(6):932-5.
3. Polayes D. Focus 1991;13(4):1302.
4. Dasso MC, Jackson RJ. Nucl Acid Res 1989;
17:3129.
Figure 1.
1 2 3 4
Comparison of pGem-luc transcript purification
methods. Transcripts were synthesized in 20 L
reactions. After DNase I treatment, RNA was
purified from the reaction mix. 500 ng of
purified RNA were run on 1% agarose/
formaldehyde gel.

The banding pattern of the 1.7 kb RNA transcripts
is identical regardless of purification method. Similar
results were obtained in the case of capped
transcript (results not shown).
Lane 1: 0.161.77 kb RNA ladder (Gibco BRL)
Lane 2: RNA purified in Microcon 30K NMWL
device
Lane 3: RNA purified by phenol extraction

P ro t o c o l s f o r Nuc l e ic Ac id s P u rif ic a t io n of I n Vi t ro Sy n t h e s i ze d mR N A
Lane 4: RNA purified by LiCl precipitation
The effect of increasing the mRNA concentration
on the translational efficiencies was examined.
At low mRNA levels, the capped luc mRNA was
translated three times more efficiently than the Figure 2.
uncapped mRNA (Figure 2). At higher mRNA levels,
140
the translation of both transcripts was comparable.
120
Similar behavior was observed with CAT mRNA3.
Even relatively high levels of mRNA did not cause 100
cpm x 104

the decrease in translational efficiencies noted by 80

other groups4. This result could be attributed in part 60
Capped pGem-luc RNA
to the lack of inhibitory contaminants in the mRNA pGem-luc RNA
40
preparation.
20
We also checked the effect of the RNA clean-up
0
method on in vitro translational efficiency. For details
0 0.5 1.0 1.5 2.0
of various procedures, see the Methods section. g RNA/50 L Reaction
RNA purified by each of the methods (1 g) was
Effect of pGem-luc RNA concentration on in vitro translation. Increasing
translated and analyzed. While there were no
amounts of uncapped Luc RNA transcripts and capped Luc RNA were used
observable differences between these RNAs by gel in translation reactions. Incorporation of 35S-methionine was determined
electrophoresis analysis (Figure 1), RNA purified by by TCA precipitation. Both RNA transcripts were purified with Microcon
ultrafiltration gave twice the translation efficiencies 30K NMWL devices.

of phenol-extracted RNA.

77
 Effect of Centrifugal Ultrafiltration on
Large Fragment DNA Integrity
Introduction again with TE buffer (to 500 L or 2 mL, depending
on the device used) and spun once more, as
Centrifugal ultrafiltration provides a fast and easy
described above. The dilution step was repeated
method for the concentration and desalting of
a third time. After the third concentration spin, the
biological molecules. Previous reports document
retentate was collected by placing the sample
the use of ultrafiltration for desalting samples of
reservoirs upside down in new vials and spinning
nucleic acids1, removing excess primers from PCR2
d sid s E f f e c t of C e n t rif u g a l U l t r af i l t r a t io n o n L a r g e F r a g m e n t D N A In te g ri t y

the units for 1 minute at 1,000 x g.

reactions3, or concentrating DNA/RNA samples
without the need to use ethanol precipitation4.
Microcon filters offer a convenient, reproducible
means for centrifugal ultrafiltration of samples from
50 L to 2 mL. They insure high sample recovery
with their patented inverted recovery spin.
The analysis of complex genomes depends on
the ability of the researcher to prepare pure, high
molecular weight DNA. When preparing high
molecular weight DNA samples for cosmid cloning
or other applications, it is important to treat the
DNA gently to avoid shearing or other damage to
the sample. As virtually all protocols for the prepa-
ration of high molecular weight DNA require, at
some point, buffer exchange and/or concentration,
centrifugal ultrafiltration can provide an efficient
alternative to standard procedures.
To be useful, centrifugation must not cause
breakage of the DNA during the ultrafiltration
procedure. This article summarizes the results of a
series of experiments designed to evaluate the effect
of g forces on various samples of high molecular
weight DNA during centrifugal ultrafiltration in
Microcon units.
cl eli ce icA ciAc
The concentrated DNA was run on a 0.9%
SeaKem GTG agarose gel (FMC) and stained
with ethidium bromide to monitor sample integrity
(Figure 1). DNA bands in lane 1 (starting material)
are indistinguishable from the bands on lanes 2 and
3, which were spun repeatedly in the ultrafiltration
device. There is no evidence that any supercoiled
DNA was converted to the relaxed or linear forms
during the concentration procedure, which would
be the case had single-strand nicks been introduced
during centrifugation.
Discrete Size Plasmids
The next set of experiments monitored the effect
of centrifugal ultrafiltration on single population,
discrete size plasmids. The plasmids used were
pBR322 (4,361 bp; New England Biolabs, Inc.),
pSPT18 (3,104 bp; Boehringer Mannheim) and
pXTl (10,400 bp; Stratagene). Samples (1 g) of
each plasmid were spun in Microcon 30K NMWL
units, as described previously. The starting material
and retentates were run on a 1% agarose gel and
stained with ethidium bromide.

Methods
Figure 1.
Nuc

1 2 3
DNA Ladder
P ro t o c o l s fo rNu

Supercoiled DNA Ladder.

As a model system to check for the introduction of
Lane 1: Starting material
single-strand nicks during the ultrafiltration spin,
Lanes 2, 3: Retentate from
supercoiled Ladder DNA (Gibco-BRL, Gaithersburg, DNA ladder spun
MD) which ranges in size from 2 to 8 kb was 3 times at 12,000 x g
in Microcon 30K
used. DNA diluted with TE buffer was loaded into NMWL device
Microcon 30K NMWL devices. The Microcon
units were spun for 7 minutes at 12,000 x g. The
concentrated DNA sample (retentate) was diluted

78
 The results are shown in Figure 2. As in the
case of the DNA Ladder, the concentrated samples Figure 2.
1 2 3 4 5 6
(lanes 2, 4 and 6) appear to be identical with their Specific plasmid DNA
corresponding starting material. Again, there is no Lane 1: pBR322 starting material
evidence of conversion of the supercoiled form to Lane 2: pBR322 spun in Microcon
the relaxed form after exposure to g forces of 30K NMWL device
5,000 x g for 90 minutes and 12,000 x g for Lane 3: pSPT18 starting material

21 minutes during the concentration spins. Lane 4: pSPT18 spun in Microcon

30K NMWL device
Genomic Size DNA Lane 5: pXT1 starting material
Lane 6: pXT1 spun in Microcon
To monitor the integrity of molecules in the size
30K NMWL device
range of genomic DNA after centrifugation, lambda

P ro t o c o l s f o r Nuc l e ic Ac id s E f f e c t of C e n t rif u g a l U l t r af i l t r a t io n o n L a r g e F r a g m e n t D N A In te g ri t y
DNA (49 kb; Boehringer Mannheim, Indianapolis,
IN) and Bsu36 l digested BacPAK6 DNA (125 kb)
was used. The samples were diluted and centri- Figure 3.
fuged as described above. The retentates and 1 2 3 4
Large fragment DNA
starting material were run on a 1% agarose gel in
Lane 1: Lambda DNA starting material
a CHEF-DR II pulsed field electrophoresis system
Lane 2: Lambda DNA spun in
Bio-Rad, Richmond, CA). Electrophoresis was Microcon 30K NMWL device
performed at 200 V at 14 C in 0.5X TBE with Lane 3: BacPAK DNA starting material
ramped pulse from 1 to 6 seconds over 14 hours. Lane 4: BacPAK DNA spun in Microcon
The results with the lambda DNA mimic those 30K NMWL device
of the other samples run in these sets of experiments.
No adverse effects are noted after spinning the
DNA at g forces up to 12,000 x g in the ultrafiltra-
tion units. However, the larger BacPAK6 DNA
does show some degradation after ultrafiltration at
both 5,000 and 12,000 x g (Figure 3, smearing
in lane 4). Although a large percentage of the
sample appears to be intact, there was loss of
integrity of the BacPAK6 DNA sample after the
concentration procedure.

Conclusions
DNA samples of up to 49 kb were concentrated
repeatedly without any loss of sample integrity.
Some loss of integrity was observed with a 125 kb
sample, although it was not complete and repre-
sents a small percentage of the total DNA in the
sample. For large fragments of DNA, centrifugal
ultrafiltration provides a fast and efficient method to
concentrate or desalt the sample. It results in high
recovery of intact product.
References
1. Takagi S, Kimura M, Katsuki M. BioTechniques
1993;14(2):21821.
2. PCR is covered by U.S. patents issued to
Hoffmann-LaRoche, Inc.
3. Sheng N, Zhang J, Whitton JL, McKee T.
BioTechniques 1993;14(5):7814.
4. Ruano G, Pagliaro EM, Schwartz TR, Lamy K,
Messina D, Gaensslen RE, Lee HC.
BioTechniques 1992;13(2):26674.

79
 DNA Extraction from Agarose Gels
with Montage Gel Extraction Kit or
Ultrafree-DA Centrifugal Filters
Introduction DNA prepared with the Ultrafree-DA centrifugal
filter requires no further purification for most applica-
The Ultrafree-DA device is designed to recover
tions, including cloning and radioisotopic or
100 to 10,000 bp DNA from agarose gel slices in
fluorescent DNA sequencing. Since agarose gel
one 10-minute spin. It consists of a pre-assembled
electrophoresis has high resolving power, the small
sample filter cup with an agarose gel nebulizer and
and large non-specific amplification products that
a microcentrifuge vial. The device uses gel com-
frequently interfere with cloning and sequencing
pression to extract DNA from the agarose.
after PCR (polymerase chain reaction) are com-
Centrifugal force collapses the gel structure, drives
pletely removed from the product.
the agarose through a small orifice in the gel
nebulizer and captures the resultant gel slurry in the
Materials
sample filter cup. As the agarose is compressed at
5,000 x g, DNA is extruded from the gels pores. Microcentrifuge
The gel matrix is retained by the microporous Pre-assembled Ultrafree-DA centrifugal filter
membrane, and the DNA passes freely through device or Montage Gel Extraction Kit
the membrane. DNA can then be recovered in
P ro t o c o l s f o r Nuc l e ic Ac id s D N A E x t r a c t io n f ro m A g a ro s e G e l s

Modified TAE* electrophoresis buffer (40 mM

the filtrate vial.
Tris-acetate, pH 8.0, 0.1 mM Na2EDTA)
The Montage Gel Extraction Kit consists of
50 Ultrafree-DA centrifugal filters as well as a SeaKem agarose (FMC BioProducts) or equivalent
modified TAE buffer that allows the casting and Long-wavelength UV lamp
running of the gel from which the DNA fragment
Scalpel or razor blade
is to be extracted.

*Modified TAE is recommended rather than TBE for the following reasons: (1) TBE buffer strongly inhibits DNA sequencing reac-
tions while modified TAE buffer does not. (2) Modified TAE has 0.1 mM Na2EDTA while regular TAE has 1.0 mM Na2EDTA. The
EDTA level at 0.1 mM Na2EDTA will not interfere with the magnesium concentration in sequencing reactions and other down-
stream enzymatic treatments, many of which are dependent on magnesium.

80
Procedure 4. Spin at 5,000 x g for 10 minutes. Centrifugation
forces the agarose through the gel nebulizer,
1. Electrophorese 30 L of PCR product or other
converting it to a fine slurry that is captured by
DNA through a <1.25% ordinary agarose gel,
the sample filter cup. Extruded DNA in electro-
prepared in modified TAE buffer with ethidium
phoresis buffer passes through the microporous
bromide (0.5 g/mL).
membrane in the sample filter cup and collects in
2. Locate the band of interest with a long wave- the filtrate vial.
length UV lamp or transilluminator. With a razor
5. DNA in the filtrate is now ready for sequencing
blade or scalpel, cut out the slice of agarose
or cloning without further purification. Discard the
(<100 L or 100 mg) containing the band
gel nebulizer and sample filter cup and store the
of interest. Trim any away excess agarose
DNA in the capped filtrate vial.
from band.
3. Place the gel slice into the gel nebulizer/sample Results
filter cup/filtrate vial assembly and seal the
Gel compression is a quick and easy technique for
device with the cap attached to vial.
recovering DNA from an agarose gel slice.

Table 1. Effect of gel disruption on typical DNA recoveries

from agarose gels
%DNA Recovered from
% DNA Recovered Gel Disrupted by Gel
DNA Size (bp) from Intact Gel Nebulizer
100 74 78

P ro t o c o l s f o r Nuc l e ic Ac id s D N A E x t r a c t io n f ro m A g a ro s e G e l s
400 39 ND
700 43 71
1000 55 77
2027 * 47
4361 14 35
9416 * 32
23130 * 29
* = Not detectable

81
 PCR Purification with
Montage PCR Filter Units
Introduction
Figure 1.
After polymerase chain reaction (PCR)1, amplified
DNA must be separated from excess reaction Primer DNA
Salt dNTP
components that can interfere with subsequent
manipulations such as cloning or sequencing.
The Montage PCR filter unit is a single-use device
that simplifies the purification of PCR products.
It concentrates amplified DNA and removes primers
and unincorporated dNTPs, while providing
excellent capacity, high recovery, and high purity.
The method is quick and highly reproducible, which
makes it ideal for processing one-up or multiple Step 1 Step 2 Step 3
samples in middle-throughput operations.

Materials
Variable speed microcentrifuge
Montage PCR filter unit
Purified water (such as Milli-Q water) or TE buffer
PCR reaction (aqueous phase)
Method
P ro t o c o l s f o r Nuc l e ic Ac id s P C R P u rif ic a t io n
Results
Montage PCR filter units were used to purify 100 L
PCR reactions according to the specified protocol.
After recovering the DNA fragments (n = 10) using
a reverse spin, the samples were separated by
agarose gel electrophoresis. Recoveries of the
various PCR products were determined by densitom-
etry (Figure 1).

1. Insert the Montage PCR sample reservoir into

The Montage PCR filter unit is a convenient
one of the two vials provided.
method for single-sample PCR purification. The
2. Fill the reservoir with 300 L purified water or high-performance device purifies PCR products
TE buffer. Add 100 L PCR reaction to the in a single centrifugation step. Purified samples
reservoir (step 1). Smaller volumes of PCR are ready for downstream applications with no
product may be used, but the volume should additional purification steps.
be adjusted to a final volume of 400 L.
3. Spin the Montage PCR device at 1000 x g References
for 15 minutes (step 2). NOTE: For optimal 1. PCR is covered by U.S. patents issued to
recovery, do not centrifuge longer than the Hoffmann-LaRoche, Inc.
specified 15 minutes or greater than 1000 x g.
4. To recover DNA, remove the sample reservoir
from filtrate collection vial and place in a
clean vial.
5. Add 20 L purified water or TE buffer to sample
reservoir.
6. Invert the reservoir and spin at 1000 x g for
2 minutes to retrieve the purified PCR (step 3).

82
Figure 2.
100%

80%
Percent Recovery

60%

40%

20%

0%
137 bp 301 bp 500 bp 657 bp 1159 bp

Purification of PCR Products Using Montage PCR Filters

Figure 3.

P ro t o c o l s f o r Nuc l e ic Ac id s P C R P u rif ic a t io n

Typical electropherogram shows an 1159 bp PCR product purified with a Montage PCR filter unit. Note the uniform signal intensity and
long read lengths. Purified samples are ready for cloning or sequencing with no additional purification steps.

83
 Enzyme Removal with Micropure-EZ
Centrifugal Filters
Introduction (Figure 1) before centrifuging again. The purified
DNA is suitable for cloning or for other enzymatic
The Micropure-EZ device is a convenient device
manipulation.
for removing enzymes from double-stranded (ds)
While most enzymes were removed by the
DNA (20 bp to >50,000 bp) solutions in a single
Micropure-EZ device (Table 1, on page 86),
centrifugation. It can be used to remove enzymes
Millipore felt it was equally important to notify
whenever heat inactivation or phenol/chloroform
customers of several enzymes that were not
extraction is impossible or impractical. After an
removed (Table 2 , on page 86). Millipore scientists
enzyme reaction, the modified DNA solution
were very careful to rule out enzyme inhibition as a
containing dilute protein is simply pipetted into a
mechanism. The sensitivity of each assay to detect
Micropure-EZ device that has been inserted into a
trace amounts of restriction enzymes in Micropure-
vial (which is supplied) and centrifuged for 1 minute
EZ filtrates was determined by carrying out a
at 12,00014,000 x g. The DNA passes freely
dilution series (this is the method used by restriction
into the vial (typically with 8590% DNA recovery)
enzyme manufacturers to estimate total activity).
while the enzyme remains bound to the proprietary
membrane in the Micropure-EZ device. Removal is
Method
defined as the complete absence of detectable
In order to ensure that each enzyme assay was
enzyme activity or, as in the isolated case of T4
detecting trace amounts of restriction enzymes in
polynucleotide kinase, inconsequential residual
Micropure-EZ filtrates, serial dilutions of Bgl I
activity (<0.08%). For simultaneous concentration
(representing 2 units, 0.4 units, 0.08 units, and
of the enzyme-free DNA, place the Micropure-EZ
0.016 units of total activity in 10 L) were carried
device into a Microcon microconcentrator
out in a reaction mix* prepared with either sterile
DI water (sdH2O) or with sdH2O that had been
P ro t o c o l s f o r Nuc l e ic Ac id s E n z y m e Re m ova l

filtered through Micropure-EZ devices and then

Figure 1. incubated. Comparison of the resultant restriction
Remove Enzyme
Restriction and digests after agarose gel electrophoreses verified
other enzymes
DNA Enzyme Salt
Micropure-EZ are absorbed by that aqueous extractables from Micropure-EZ devices
Micropure-EZ.
dsDNA passes did not inhibit the enzyme. Also, these standard
freely. curves determined the sensitivity of the enzyme
Remove Enzyme
Restriction and assay (i.e., the theoretical amount of residual
other enzymes Concentrate DNA
DNA is retained by enzyme activity that could be detected, if present).
Micropure-EZ are absorbed by Microcon
Micropure-EZ. Assembly ultrafilter in Microcon. To stress test Micropure-EZ devices sufficiently
dsDNA passes Salts pass freely.
freely. with enzymes and DNA, several extreme operating
Vial conditions were used consistently. Micropure-EZ
Recover devices were challenged with 50 units of Bgl I in
85% DNA
the presence of decoy DNA (1 g of pBR322) and
5 g of bovine serum albumin (BSA). Devices were
spun at 14,000 x g for 30 seconds. The filtrates
30 seconds 3 minutes were then assayed for residual enzyme activity by
Operation of Micropure-EZ device adding 1 L of pUC19 DNA and 0.5 L of 100X
BSA to the filtrate, mixing thoroughly and incubating

*15.8 L of sdH2O, 2 L of 10X NEBuffer 2, 0.2 L of 100X BSA, and 2 L of Bgl I (10 units/L)

84
at 37 C for 1 hour. After a brief centrifugation to
collect the condensate at the bottom of the micro-
centrifuge tubes, 1 L of 0.5 M disodium EDTA
and 10 L of 5X loading buffer were added to
stop the reaction.
The negative control was a portion of DNA
master mix. Control digests of pBR322 DNA and
pUC19 DNA were carried out separately.
Results and Discussion
The Bgl I standard curve made with the Micropure-
EZ-filtered sdH2O was indistinguishable from
the standard curve made with untreated sdH2O.
The plasmid DNA (1 g of pUC19 and 1 g of
pBR322) was cut to completion by 2 units of Bgl I
after one hour at 37 C (Figure 2, lanes 1 and 6).
None of the enzymes shown in Tables 1 or 2 was
measurably inhibited by Micropure-EZ-filtered
sdH2O. The standard curves indicated that as little
as 0.08 units of Bgl I could be detected by this
assay (lanes 3 and 8).
The devices removed the 50 units of Bgl I as
evidenced by an absence of detectable activity in
the filtrates. In the lanes corresponding to the Bgl I
challenged Micropure-EZ inserts, some activity was
observed against the decoy pBR322 DNA, as
expected during its brief exposure to Bgl I (particu-
larly evident in lane 14). However, the pUC19,
which was added directly to the filtrate and
incubated, appeared completely intact (lanes
1114). Since the standard curve indicated that
0.08 units would be detected if it were present,
we calculated that at least 99.8% of Bgl I was
removed and/or inactivated. Removal, rather than
inactivation, is the probable mechanism by which
Micropure-EZ devices operate, since in separate
experiments we were unable to detect any enzyme
(irrespective of activity) in Micropure-EZ filtrates
using very sensitive HPLC methods (data not shown).
Summary
Enzyme removal and excellent DNA recovery are
accomplished with Micropure-EZ devices in a single
60-second spin without any pre-wetting, binding,
washing or elution step. Cumbersome phenol/
chloroform extraction and the associated hazardous
waste accumulation are avoided. An additional line
of evidence for the suitability of Micropure-EZ
devices in molecular biology applications is that
restriction digest purified with Micropure-EZ devices
alone are readily ligated and cloned into plasmid
DNA (M.H.A.L., unpublished observations).
Micropure-EZ devices used alone or with Microcon
devices permit rapid and efficient sequential
enzymatic DNA manipulations.

P ro t o c o l s f o r Nuc l e ic Ac id s E n z y m e Re m ova l
Figure 2.
Analytical agarose gel, demonstrating the detection limits for Bgl I and Bgl I removal by Micropure-EZ devices.

Lanes 14: Serial dilutions of BgI I representing 2 units, 0.4 units, 0.08 units, and 0.016 units of activity (respectively)
against the DNA master mix prepared in non-filtered sdH2O
Lane 5: Undigested DNA master mix used as substrate for the standard curve containing pUC19 and pBR322 DNA
Lanes 69: Inhibition control: serial dilutions of BgI I representing 2 units, 0.,4 units, 0.08 units, and 0.016 units of activity (respectively)
against the DNA master mix prepared in Micropure-EZ-filtered sdH2O
Lane 10: Molecular weight standard: 1 kb DNA ladder (Gibco-BRL, Gaithersburg, MD)
Lanes 1114: Filtrates incubated with pUC19 DNA after challenging Micropure-EZ devices with 50 units of BgI I, BSA and
decoy pBR322 DNA
Lane 15: Uncut pBR322
Lane 16: BgI Icut pBR322
Lane 17: Uncut pUC19
Lane 18: BgI Icut pUC19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

85
 Table 1. Enzymes removed by Micropure-EZ devices

Enzyme Challenge Enzyme Challenge Enzyme Challenge

AMV reverse transcriptase 50 U
Calf intestinal alkaline 10 U
phosphatase
DNase I (bovine pancreas) 10 U
Exonuclease III (E. coli) 100 U
MMLV reverse 600 U
transcriptase
Mung bean nuclease 50 U
Proteinase K (Amresco) 5 g
T4 DNA ligase 2,000 U*
T4 DNA polymerase 15 U
T4 polynucleotide kinase** 50 U
Taq DNA polymerase 5U
Terminal deoxynucleotidyl 45 U
transferase
Acc I 50 U
Apa I 100 U
BamH I 100 U
Bcl I 50 U
Bgl I 50 U
BsiW I 70 U
BssH II 20 U
BstN I 50 U
Dpn I 100 U
EcoR I 100 U
Hae III 100 U
Hinc II 50 U
Hind III 100 U
Hpa I 25 U
Kpn I 50 U
Mbo I 25 U
Mlu I 50 U
Nco I 50 U
Nde I 100 U
NgoM I 50 U
Nhe I 25 U
Not I 50 U
Nru I 50 U
Pst I 100 U
Sac I 100 U
Sac II 100 U
Sal I 100 U
Sca I 50 U
Sph I 25 U
Sst I 50 U
Xho I 100 U
In tests, all enzymes were removed
from solutions containing DNA or
RNA (in the case of RNase A). Five
g of bovine serum albumin were
also present during removal of restric-
tion enzymes. Removal was indicated
by undetectable or inconsequential
enzyme activity in filtrate (<0.08%
residual in the case of T4 polynucleo-
tide kinase).

*New England Biolabs unit definition

**T4 polynucelotide kinase is not recommended with oligonucleotides (dsDNA only). The results suggest that
this kinase mediates the binding of oligos to the membrane in Micropure-EZ device, causing oligo loss.

Table 2. Enzymes not removed by Micropure-EZ devices

d sid s E n z y m e Re m ova l

Enzyme Challenge Enzyme Challenge

Bacterial alkaline 0.6 U Eae I 15 U
phosphatase EcoR V 50 U
DNA polymerase I 20 U Hinf I 50 U
(Klenow)
Msp I 100 U
Exonuclease I 50 U
Pvu I 25 U
Pfu DNA polymerase 2.5 U
Pvu II 50 U
Vent DNA polymerase 4U
Sau3A I 20 U
cl eli ce icA ciAc

Shrimp alkaline 1U
Sfi I 50 U
phosphatase
Sma I 25 U
Nuc

RNase A (bovine 1 g
P ro t o c o l s fo rNu

pancreas)
ApaL I 10 U
Bgl II 50 U
BsoB I 50 U
Cla I 25 U
Xba I 50 U
In tests, Micropure-EZ devices did
not remove the indicated number of
units of the listed enzymes. It may be
effective in removing a lower number
of units.

*T4 RNA ligase is not recommended. Results suggest this ligase mediates binding of
nucleic acids to the membrane in Micropure-EZ devices, causing sample loss.

86
Protocols for
Concentration of Bacteriophage
Using Ultrafiltration . . . . . . . . . . . . . . . . . . . . . 88

Virus Concentration
Concentration of Animal Viruses
Using Ultrafiltration . . . . . . . . . . . . . . . . . . . . . 91

P ro t o c o l s f o r V iru s C ONC EN T RAT ION

87
 Concentration of Bacteriophage
Using Ultrafiltration
Introduction Ultrafiltration is an alternative method to achieve
high bacteriophage titer. When complete purifica-
Bacteriophages were first discovered nearly a
tion from cell and media proteins is not required,
century ago1. Since then, phage biology has
phage can be quickly concentrated using centrifu-
drawn researchers attention because of their
gal ultrafiltration9. Ultrafiltration can also be used to
potent bactericidal capacity that can be used to
concentrate phage from environmental sources10.
treat human infections2. Bacteriophage and animal
For larger volume lysates, recirculating tangential
viruses have been successfully utilized as DNA
flow filtration (TFF) is a better choice11,12. Correct
carriers, or cloning vectors. The advantage to
membrane material, pore size, centrifugation speed
P ro t o c o l s f o r V iru s C ONC EN T RAT ION C o n ce n t r a t io n of B a c te rio p h a g e U s i n g U l t r af i l t ra t i o n

lambda-based vectors is that they can carry

fragments of DNA up to 25,000 bp. Another
application, phage display, is a novel genetic
selection system for cloning proteins from cDNA
libraries. In addition, there are many similarities
between bacteriophages and animal cell viruses.
Thus, bacteriophages can be viewed as model
systems for animal cell viruses.
The standard bacteriophage propagation
protocol normally involves infection of the host
bacterial strain, 1216 hours of bacterial growth,
followed by the phage recovery from the superna-
tant. Purification of phage usually includes a
density gradient centrifugation, followed by
extensive dialysis3,4. Phage titers are then deter-
mined by plaque assays.
For some experiments, high titer bacteriophage
stock is desired. Traditionally, phage particles were
concentrated by polyeteylene glycol (PEG) and
dextran sulfate precipitation, precipitation by acid,
or differential or equilibrium centrifugation5. Recently,
different modifications of the Sambrook method are
being used6. Phage is concentrated by precipitation
with sodium chloride and PEG and resuspended
to the desired titer. The process requires 612
hours of incubation with PEG and a lengthy pellet
resuspension process to achieve high particle
recovery 7,8.
and final retentate volume are critical to obtaining
high phage recovery and maintaining its infectivity.
This protocol describes the use of Millipore
ultrafiltration devices to concentrate Pseudomonas
pseudoalcaligenes bacteriophage phi-6, an
enveloped dsRNA virus. The virions are spherical
and 86 nm in diameter.
Method
Phi-6 production
Note: All manipulations should be performed using
aseptic technique in a laminar flow hood.
1. Grow fresh culture of Pseudomonas pseudoalca-
ligenes until it reaches the desired optical density
of 0.1 absorbance units at 600 nm.
2. Inoculate the culture with enough phi-6 to
achieve a multiplicity of infection (MOI) of 0.5.
3. Return the phi-6 containing Pseudomonas
pseudoalcaligenes culture to the platform
shaker and incubate at 26 C, 125 RPM,
for 1824 hours.
4. After the overnight incubation, aliquot the culture
into four 250 mL centrifuge bottles.
5. Centrifuge the bottles for 30 minutes at
16,000 x g.
6. Remove the supernatant and transfer to a
sterile container.

88
Concentration using Amicon Ultra-4 or Ultra-15
devices with 50 kDa NMWL membrane
1. Clarify phi-6 supernatant using a Stericup
filter unit (Millipore cat. no. SCGP U05 RE),
a Steriflip filter unit (Millipore cat. no.
SCGP 005 25), or a Millex-GP syringe filter
(Millipore cat. no. SLGP 033 RB) with low-
binding 0.22 m PES membrane.
2. Load appropriate volume of clarified phi-6
supernatant (4 or 15 mL) into an Amicon Ultra-4
device (Millipore cat. no. UFC8 050 24) or
an Amicon Ultra-15 device (Millipore cat. no.
UFC9 050 24) containing 50,000 NMWL
regenerated cellulose membrane.
3. Centrifuge the device (4 or 15 mL) for 15
minutes at 1500 g.
4. The recoverable volume should be 80100 L
for Amicon Ultra-4 devices and 350500 L
for Amicon Ultra-15 devices.
Note: Avoid concentrating samples below
recommended volumes as this may lead to
decreased phage recovery.
Concentration using Pellicon XL50 device
with PLCTK membrane and Millipores
Labscale TFF system
Note: The Labscale TFF system (Millipore cat. no.
XX42 LSS 13) is not required to use Pellicon XL50.
A peristaltic pump may alternatively be used
(see Millipore user guide P60085).
1. Perform flush, integrity, and permeability tests
on Pellicon XL50 device with PLCTK membrane
(Millipore cat. no. PXCO 30C 50) as indicated
in the Pellicon XL50 User Guide.
2. Remove reservoir cover and load up to 500 mL
phi-6 supernatant into reservoir of Labscale
TFF system.
3. Ensure vent port is open by removing plug from
VENT port and leaving open or installing a
Millex filter.
4. Open tank outlet valve.
5. Turn on pump and increase speed until feed
pressure reaches 20 psi.
Note: Check system connections for leaks.
Tighten connections as required.
6. Set retentate pressure by slowly adjusting
retentate valve clockwise until retentate
pressure is 10 psi.
7. Adjust pump speed and retentate valve
restriction to achieve desired feed pressure
of 30 psi and retentate pressure of 10 psi.
Note: Do not exceed feed pressure of 60 psi.
8. Filter the solution until the desired concentration
or volume is obtained.
9. Stop pump.
10. Disconnect pump outlet tubing from pump
outlet port and place in product recovery
collection vessel.
P ro t o c o l s f o r V iru s C ONC EN T RAT ION C o n ce n t r a t io n of B a c te rio p h a g e U s i n g U l t r af i l t ra t i o n
11. Disconnect retentate tubing from retentate
in port. Fluid should now drain by gravity.
If additional drainage is required, a syringe
can be placed on the end of the retentate
tube and fluid can be blown down.
12. Replace retentate tubing in retentate port.
Reconnect pump outlet tubing.
13. Disconnect FEED IN tubing and place in
collection vessel. Open tank outlet valve,
turn pump speed up to drain reservoir.
14. Reconnect the pump outlet tubing to the
FEED IN port.
Results
Millipore ultrafiltration devices with Ultracel regener-
ated cellulose membrane offer low non-specific
binding and high retentate recovery. The vertical
membrane reduces concentration polarization for
ultra-fast concentration times and is especially
beneficial for turbid and viscous solutions.
As shown in the protocol above, phage titer
can be increased up to 30-fold in 15 minutes by
centrifugal ultrafiltration for under 15 mL volumes.
Amicon Ultra-4 and Ultra-15 devices with 50 kDa
NMWL membranes can be used successfully to
concentrate the phage phi-6 out of bacterial culture
supernatant. The same devices with 100 kDa
NMWL membranes are not recommended because
they lead to a low virus recovery and allow virus to
break through into the filtrate.
89
 For large volume concentration, tangential flow
filtration (TFF) should be used. Table 1 shows phage
concentration results using a Pellicon XL50 cassette
with the Labscale TFF system. The Pellicon XL50
cassette couples Millipores ultrafiltration membranes
with the linearly scalable TFF cassette for processing
501000 mL volumes. The Ultracel membrane with
30 kDa NMWL is recommended. Twenty-fold
concentration of phage solution is usually achieved
in 3050 minutes.
5. Bachrach U, Friedmann A. Appl Microbiol
1971;22(4): 706-715.
6. Sambrook J, Fritsch EF, Maniatis T. Molecular
cloning: a laboratory manual, 2nd ed. Cold
Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. 1989
7. Bahns JT, Liu CM, Chen L. Protein Science
2004;13:2578-2587.
8. Gill JJ, et al. Chemother 2006;50(9):
2912-2918.

References 9. Meile L, Abendschein P, Leisinger T.

J Bacteriol 1990;172(6):3507-3508.
1. Pennazio S. Riv Biol 2006;99(1):103-29.
10. Middelboe M, et al. Aquat Microb Ecol
2. Fischetti VA, Nelson D, Schuch R. Nature
2003;33:1-10.
P ro t o c o l s f o r V iru s C ONC EN T RAT ION C o n ce n t r a t io n of B a c te rio p h a g e U s i n g U l t r af i l t ra t i o n

Biotechnology 2006;24:1508-1511.
11. Rembhotkar GW, Khatri GS. Anal Biochem
3. Hansen MR, Mueller L, Pardi A. Nature
1989;176(2):373-4.
Structural Biology 1998;5:1065-1074.
12. Alonso MC, Rodriguez J, Borrego J. J Internatl
4. Zhilenkov EL, et al. Virology Journal
Microbiol 1999;2:227-232.
2006;3:50-55.

Table 1. Bacteriophage phi-6 concentration using Millipore ultrafiltration devices

Amicon Ultra-4, Amicon Ultra-4, Amicon Ultra-15, Pellicon XL50 TFF,
100 kDa 50 kDa 50 kDa 30 kDa
Initial volume (mL) 4 4 15 500
Initial viral titer, pFU/mL 2.3 x 10 10
2 x 10 10
2.02 x 10 10
1.9 x 107
Final volume (mL) 0.1 0.5 0.5 25
Final viral titer, pFU/mL 2.06 x 10 11
3.7 x 10 11
7.8 x 10 11
5.6 x 108
Concentration factor 40 8 30 20
Phage recovery (%)* 22 >100 >100 >100
Phage in the filtrate, pFU/mL 2.3 x 105 <100 <100 7 x 103
*We consistently observe higher than 100% recovery after phage concentration. This can be explained by increased infectivity of phage particles at
higher concentrations and agrees with other published data12.

90
Concentration of Animal Viruses
Using Ultrafiltration
Introduction
Despite the rapid progress in genomics, the biologi-
cal function of the majority of the human genes is
still unknown. The identification of gene function is
an increasingly relevant issue, especially in the
search for new targets for improved human disease
therapy. Functional genomics approaches, which
aim at the identification of genes via phenotypes
induced in biological systems, require measurement
of gene function on the genomic scale in cell-based
assays. cDNA expression libraries representing the
population of expressed genes in a given cell/tissue
type have classically been cloned into plasmids,
which can be introduced into cells by transient
transfection. However, transfection efficiency varies
widely between cell types. Therefore, a variety of
virus-derived vector systems have been developed
for improved cDNA transduction, expression, and
screening in mammalian cells. These systems include
simian virus 40 replicons in COS cells1,2 and
cDNA cloning and expression vectors derived
from retrovirus3,4 baculovirus5, alphavirus6, human
immunodeficiency virus7, vaccinia virus8, adenovirus9
Figure 1. Primary culture of rat cortical
neurons infected with lentivirus
For virus propagation, HEK-293T cells were trans-
fected with helper plasmids and lentiviral plasmid
expressing copGFP (System Bioscience). 48 hours
after transfection viral supernatant was collected
and 10 mL was concentrated using Amicon Ultra-15
100 kDa MWCO membrane to 200 L. 50 L of
concentrated viral stock was used for infection of
10E6 cells.
Data courtesy of Drs. Leila Aminova and Ambreena Siddiq,
Neuroprotection Laboratory of Beth Israel Medical Center.
and adeno-associated virus (AAV)10. Challenges
associated with the use of viral vectors in clinical
trials include production of sufficient quantities of
clinical grade material and maintaining biosafety of
the viral vector. Production and purification methods
for viral vectors vary both in terms of methodology
and virus yield and quality.
Generation and purification of viral vectors can
be labor intensive, costly and may require special
P ro t o c o l s f o r V iru s C ONC EN T RAT ION C o n ce n t r a t io n of A n i m a l V i ru s e s U s i n g U l t r af i l t ra t i o n
equipment (such as ultracentrifuges). The standard
method for purification of adenovirus, lentivirus
and AAV uses density gradient ultracentrifugation
followed by extensive dialysis. More recently,
column or membrane chromatographies have
become the methods of choice for adenovirus
purification11,12. The yield of virus normally is
determined by plaque assay, TCID50 assay and
more recently by ELISA assay. For most applications,
recombinant virus must be purified to a high titer.
In order to achieve high titer stock, it is usually
necessary to concentrate purified virus particles.
This Protocol describes the use of Millipore centrifu-
gal ultrafiltration devices for concentration of three
most common virus types: lentivirus, adenovirus and
adeno-associated virus (AAV).
Method
1. Packaging cells: Host cells are plated to achieve
8085% confluence
2. Transfection: Cells are either transfected with
recombinant plasmid(s), or infected with viable
virus particles.
3. Harvesting virus: Depending on the virus, either
cell supernatant or pelleted cells, or both, are
used to purify the recombinant virions. Three to
four rounds of freeze/thaw cycles are usually
applied to release virus from cells. Cell debris is
then separated from cell lysate by centrifugation.
4. Virus purification: Virus is purified from cell
lysate by density gradient ultracentrifugation or
column/membrane chromatography. In some
cases, affinity chromatography with heparin
or mucin sepharose13,14,15 can be used.
91
 Purified virus solutions obtained via these methods
are then buffer exchanged using either passive
dialysis or diafiltration into an appropriate storage
solution.
Virus Concentration
In order to achieve a high titer of viral stock, the
purified virus can be concentrated using centrifugal
ultrafiltration. The correct choice of device, mem-
brane material, MWCO, centrifugation speed,
centrifugation time and buffer composition are
critical for high recovery of infective viral particles.
Amicon Ultra centrifugal ultrafiltration devices with
a 50 KDa NMWL Ultracel regenerated cellulose
membrane have been demonstrated to successfully
P ro t o c o l s f o r V iru s C ONC EN T RAT ION C o n ce n t r a t io n of A n i m a l V i ru s e s U s i n g U l t r af i l t ra t i o n
Table 1 shows typical results of concentration of
adenovirus, lentivirus and AAV from both crude cell
lysate and virus purified by chromatography.
It is important to remember that most of the viruses
cannot be efficiently concentrated and stored in
low salt buffers. Centrifugal concentration of the
virus in <200 mM salt solutions will lead to virus
aggregation and decreased infectivity. An example
of a recommended storage solution is 20 mM Tris,
pH 8.0, 250 mM NaCl, 10 mM MgCl2,
5% sorbitol and 0.001% PF68 (pluronic acid)16.
Concentrated viral stock can also be filter
sterilized through 0.22 m filters. For example,
Steriflip-GP filter units (cat. no. SCGP 005 25)

can be used for volumes of 50 mL or less or

concentrate adenovirus and AAV solutions. For Ultrafree-MC GV (cat. no. UFC3 0GV 0S)
lentivirus concentration, devices with both 50 and can be used for volumes of <0.5 mL.
100 kDa membranes can be used.

Table 1. Concentration of virus using Millipore ultrafiltration devices

Starting Starting Final Spin Time at Recovery Concentration
Starting solution Device Virus Titer Volume (mL) volume (L) 1500 x g (min) (%)* Factor
Adenovirus in crude Amicon Ultra-4, 1 x 108 4 150 20 >100 27X
cell lysate 50 kDa MW
Adenovirus purified, Amicon Ultra-4, 1 x 108 4 80 10 85 45X
in 1M salt buffer 50 kDa MW
AAV in crude Amicon Ultra-4, 2 x 107 4 200 35 50 20X
cell lysate 50 kDa MW
AAV in crude Amicon Ultra-15, 2 x 107 10 400 45 76 25X
cell lysate, diluted 50 kDa MW
in 100 mM Tris,
200 mM NaCl buffer
AAV in crude Amicon Ultra-4, 0.5 x 107 2 200 20 100 20X
cell lysate, diluted 50 kDa MW
in 100 mM Tris,
200 mM NaCl buffer
Lentivirus in cell Amicon Ultra-4, 9 x 104 4 40 30 >100 100X
culture supernatant 100 kDa MW
Lentivirus in cell Amicon Ultra-4, 9 x 104 4 40 30 93 100X
culture supernatant 50 kDa MW
Lentivirus in cell Centricon 70 Plus, 8 x 103 65 800 30 81 80X
culture supernatant 100 KDa MW
*We do not recommend concentrating the virus to higher than 1013 particles/mL due to potential virus aggregation17.

92
References
1. Gearing DP, et al. EMBO J 1989;8:3667.
2. Yokota T, et al. Proc Natl Acad Sci 1984;81:
1070.
3. Kitamura T, et al. Proc Natl Acad Sci 1995;
92:9146.
4. Shaughnessy L, et al. J Mol Neurosci 2004;
24:23.
5. Granziero L, et al. J Immunol Methods 1997;
203:131.
6. Koller D, et al. Nat Biotechnol 2001;19:851.
7. Van Maanen M, et al. Mol Ther 2003;8:167.
8. Smith ES, et al. Methods Mol Biol 2004;

P ro t o c o l s f o r V iru s C ONC EN T RAT ION C o n ce n t r a t io n of A n i m a l V i ru s e s U s i n g U l t r af i l t ra t i o n

269:65.
9. Elahi SM, et al. Gene Ther 2002;9:1238.
10. Choi VW, et al. Curr Gene Ther 2005;
5:299.
11. Blanche F, et al. Gene Therapy 2000;7:
1055-1062.
12. Shabram PW, et al. Hum Gene Therapy 1997;
9:453-465.
13. Clark KR, et al. Hum Gene Therapy 1999;
10:1031-1039.
14. Zolotukhin S, et al. Gene Therapy 1999;6:
973-985.
15. Auricchio A, et al. Hum Gene Therapy 2001;
12:71-76.
16. Alejandra E, Arbetman, AE, et al. Journal of
Virology 2005;79(24):15238-15245.
17. Galdiero F. Arch Virol. 1979;59(1-2):99-105.

93
 High Throughput Applications
The MultiScreen filter plate with Ultracel-10
membrane provides a new method for high
throughput sample prep. The ultrafiltration-based
filter plate is designed for use with centrifugation
and is compatible with standard microtiter plates,
instrumentation, and liquid handling equipment.
All of the publications summarized below
can be supplied by your local Millipore office or
downloaded from www.millipore.com/ultracel.
Nucleic Acid Purification and Concentration
AN1040EN00
The MultiScreen Filter Plate with Ultracel-10
membrane can be used to purify and concentrate
single-stranded oligonucleotides and double-
stranded DNA fragments, as well as plasmid
and genomic DNA.
Purification of Serum Peptides for
Biomarkers Research
AN2010EN00
This study is a high throughput version of the
application note on page 42 of the Protocols
section of this handbook.
Enzymatic Activity Recovery
AN2011EN00
This study demonstrates the use of the MultiScreen
plate with Ultracel-10 membrane for concentrating
alkaline phosphatase without loss of biological
activity and with high reproducibility across
the plate.

Concentration of Proteins in Cell Lysate

AN1424EN00
The MultiScreen filter plate with Ultracel-10
membrane can be used to concentrate whole
cell lysates without loss of protein and with high
reproducibility across the plate. Applications include
parallel protein purification, protein concentration
and buffer exchange in cell lysates for subsequent
separation or assaying.
App e nd i x H i g h T h ro u g h p u t A p p l i c a t io n s

MultiScreen Filter Plate with Ultracel-10 ultrafiltration

membrane

94
Glossary
Asymmetric Membrane1
Membrane constituted of two or more
structural planes of non-identical
morphologies.
Batch Process
A fixed volume of solution contained in a
tank to which the concentrate is returned
during the process.
Composite Membrane1
Membrane having chemically or structurally
distinct layers.
Concentration Polarization
Accumulation of rejected solute on the
membrane surface. Depends on interactions
of pressure, viscosity, crossflow (tangential)
velocity, fluid flow conditions, flow channel
conditions and temperatures.
Crossflow (Tangential Flow)
Solution flows across (tangential to) a
membrane surface. Facilitates back diffusion
of solute from that surface into the bulk
solution, counteracting concentration
polarization.
Diafiltration
Removal of small components from the
retained species during ultrafiltration by
adding water or buffer solution to the
retentate. See page 7 for further discussion.
Dialysis
Diffusive transport of ions or other small
molecules through a membrane barrier
that contacts solvent on both sides. containing solvent and solutes not retained
Downstream1
Side of a membrane from which the
permeate emerges.
Feed (Sample)
The starting solution (sometimes the solution
remaining upstream of the membrane).
Fluid Velocity
The flow rate of solution across the
membrane surface in cross (tangential) flow.
Related to hydraulic pressure drop.
Flux
The filtration rate through the membrane
per unit area.
Fouling Reverse Osmosis1
Irreversible decline in membrane flux due to Liquid-phase pressure-driven separation
deposition and accumulation of submicron process in which applied transmembrane
particles and solutes on the membrane pressure causes selective movement of
surface. Also, crystallization and precipita- solvent against its osmotic pressure
tion of small solutes on the surface and in difference.
the pores of the membrane. Not to be
Tangential Flow Filtration (TFF)
confused with concentration polarization.
Flow through a membrane device in which
Membrane1 the fluid on the upstream side moves
Structure having lateral dimensions much parallel to the membrane surface.
greater than its thickness, through which
Transmembrane Pressure (TMP)
mass transfer may occur under a variety
The driving force in ultrafiltration. In a stirred
of driving forces.
cell, equivalent to gas pressure. In centrifu-
Molecular Weight Cut-off (MWCO) gal devices, it is related to g-force. In a
See Nominal Molecular Weight Limit. flowing system, TMP decreases as the
stream moves from inlet to outlet. Average
Nanofiltration1
TMP = [(Pin + Pout)/2] Ppermeate
Pressure-driven membrane-based separation
process in which particles and dissolved Ultrafiltration1
molecules smaller than about 2 nm are Pressure-driven membrane-based separation
rejected. process in which particles and dissolved
molecules smaller than 0.1 m and larger
Nucleotide Cut-off (NCO)
than about 2 nm are rejected.
The number of nucleotides in a DNA
fragment (single- or double-stranded) at Yield
which 90% of the fragment is retained by Amount of species recovered at the end of
the membrane. the process as a percentage of the amount
present in the feed solution.
Nominal Molecular Weight Limit (NMWL)
The molecular weight at which at least 90%
of a globular solute of that MW is retained References
by the membrane. 1. Terminology for membranes and
Permeate (Filtrate, Ultrafiltrate) membrane processes (IUPAC
The solution passing through the membrane, Recommendations 1996). IUPAC,
Journal
by the membrane. of Membrane Science.1996;120(1):
14959.
Plugging
Accumulation of debris on the fluid flow
path, restricting or blocking flow.
Rejection App e nd i x G l o s s a r y
The fraction of solute held back by the
membrane. Can be measured at any point
in the process or averaged over the run.
Retentate (Reject Stream, Concentrate)
The solution containing the retained
(rejected) species.
Retention Factor1 (rF)
Parameter defined as one minus the ratio
of permeate concentration to the retentate
concentration of a component. Note:
rF = 1 [p]/[r] where [p] = concentration
of solute in permeate, and [r] = concentra-
tion of solute in retentate.
95
 Acknowledgements
Millipore also wishes to acknowledge the following
individuals for their contributions:

Eduardo Vottero
University of British Columbia

Peter A. Lemaire and Dr. James Cole

University of Connecticut

Gary Smejkal
Proteome Systems
Woburn, MA

Leonid Kryazhev
Genome Quebec
Montreal, Canada

Mathew L. Thakur
Thomas Jefferson University Hospital
Philadelphia, PA

Mark Merchant
Helena Laboratories
Beaumont, TX

Department of Forensic Biology

App e nd i x

New York City

Patrick O. Brown, Max Diehn, Ash Alizadeh

Stanford University School of Medicine

Dr. Sophia N. Karagiannis

GKT School of Biomedical Sciences
London

96
Patent
PCR is covered by US patents issued to Hoffmann-LaRoche, Inc.

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