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Ultrafiltration
applications, including blotting, DNA
and Upstate, which are now part of Millipore.
sequencing sample preparation, sterile
Visit www.millipore.com/immunodetection.
filtration, and MS sample prep. To contact a
Specialist, call your local office or submit a
question at www.millipore.com/techservice.
To access our library of frequently asked
questions, go to www.millipore.com/faqs.
Application and Product Guide
1
2
Ta bl e o f C o n t e n t s
Overview of Membrane Processes . . . . . . . . . . . . . . . . . . . 4
Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
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
Figure 1. Comparison of ultrafiltration with other commonly used membrane separation techniques
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.
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
Figure 2. Ultrafiltration membranes vs. traditional microporous membranes
Ultrafiltration membranes Microporous membranes are
generally have two distinct generally rigid, continuous
layers: a thin (0.11.5 m), meshes of polymeric material
dense skin with a pore diameter with defined pore sizes. They
of 10 400 and a more are used to retain bacteria,
porous substructure. Any species colloids and particulates.
capable of passing through the Species are either retained
pores of the skin (whose size is on the membrane surface or
precisely controlled in manufac- trapped in its substructure.
ture) can therefore freely pass
Cross-section of ultrafiltration Cross-section of traditional micro-
the membrane.
membrane with skin and porous porous membrane with uniform
substructure. pore structure from top to bottom.
5
Recovery
The ultimate aim of ultrafiltration is to maximize When using membrane ultrafiltration for sample
recovery of solutes of interest, but there are many concentration or desalting, care must be taken
membrane characteristics that affect that goal. to select a membrane (or device) with a NMWL
Factors affecting recovery include: appropriate for the application. Because there
Nominal molecular weight limit (NMWL)/ are several considerations in determining whether
nucleotide cut-off (NCO) a given solute will or will not be retained by a
membrane of a specific cut-off, it is best to choose
Retention
a device with cut-off at about one half of the
Concentration polarization molecular weight of the protein to be concentrated.
Flux This maximizes protein recovery and minimizes
filtration time.
Nominal Molecular Weight Limit
A microfiltration membranes pore size rating,
Nucleotide Cut-off (NCO)
typically given as a micron value, indicates that For most membranes, the NMWL is determined
particles larger than the rating will be retained. experimentally under a standard set of operating
Ultrafiltration membranes are rated according to conditions. These analyses typically employ purified
the nominal molecular weight limit (NMWL), also globular proteins to serve as markers or indicators
sometimes referred to as molecular weight cut-off of the retention characteristics of an ultrafiltration
(MWCO). The NMWL indicates that most dissolved membrane. Although this approach is useful for
macromolecules with molecular weights higher than choosing the appropriate NMWL for most protein
the NMWL will be retained. research applications, selection of a membrane
An ultrafiltration membrane with a stated NMWL with an appropriate NMWL membrane for nucleic
should retain (reject) at least 90% of a globular acid or polysaccharide purification is considerably
solute of that molecular weight in daltons. However, more complex. By virtue of the rod-like three-
for a wider safety margin, the selected cut-off dimensional structures of these molecules, these
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 types of molecules require a tighter membrane
solute to be retained. When solutes are to be (with a smaller cut-off) than do globular proteins of
exchanged, the cut-off should be substantially the same molecular weight. It is therefore convenient
above that of the passing solute. A lower NMWL to consider the membrane retention characteristics
increases rejection but decreases the filtration rate of nucleic acids as being related to their length
for the same membrane material. (in nucleotides) rather than their molecular weight.
Retention and product recovery are a function Complicating matters even further are several
of a variety of other factors, including the molecular additional factors that affect the recovery of nucleic
shape and size of the molecule; electrical charac- acid fragments from a membrane of a given
teristics; sample concentration and composition; NMWL. These factors include: the strandedness
operating conditions; and device or system of the DNA or RNA molecule; whether the DNA is
configuration. Two membranes may have the linear, relaxed or supercoiled (for plasmid); the ionic
same NMWL but will exhibit different retention of strength of the solvent; the velocity of the process
molecules within a relatively narrow range of sizes. stream over the membrane; and the nature of the
In addition, slender, linear molecules (e.g., nucleic driving force. The overall effect is that optimal
acids) may find their way through pores that will nucleic acid recovery is achieved in low salt buffers
retain a globular species of the same weight. run under conditions of relatively low velocity
Retention can also be affected by hydration with (e.g., low vacuum pressure or low g-force).
counter-ions. Nevertheless, NMWL has proven The membrane and protocol developed for the
to be an effective general indicator of membrane Montage PCR centrifugal filter device takes into
performance for globular proteins. account these conditions in order to provide for high
6
recovery of small PCR products (e.g., ~150 base of the NMWL and the size of the molecule but
pairs [bp]) as quickly as possible. For purifications Millipores recommendation is designed to provide
that are driven by vacuum, significantly tighter maximum recovery. Please see additional information
membranes are typically required to obtain regarding membrane NMWL selection on page 14.
optimal recovery.
If the DNA sample is in the presence of high salt Concentration Polarization
(or the device is run at a higher-than-recommended Another factor affecting the retention characteristics
g-force), a significantly reduced DNA recovery is the potential for membrane fouling, or concentra-
may be observed. Under these conditions, higher tion polarization. This occurs when there is an
DNA or RNA recovery can be achieved by using a accumulation of the retained solute on the surface
tighter membrane. However, it will take significantly of the membrane. At high concentrations, a gel
longer to complete the purification. For applications layer forms that can act as a secondary membrane
such as PCR where removal of unincorporated (Figure 3). This may interfere with passage of the
single-stranded primers from double-stranded DNA molecules through the membrane and can adversely
fragments is required, the molecular weights of affect the flow rate. In addition, pH, buffer compo-
the primer and DNA fragment should differ by at nents, and concentration can result in a protein
least an order of magnitude for efficient separation. behaving in an anomalous manner in terms of its
Millipore offers devices that are specifically retention or passage by UF membranes.
designed for separating and concentrating During concentration polarization, the gel layer
genomic DNA and PCR products by ultrafiltration. on the membrane surface superimposes its own
rejection characteristics on those of the membrane.
Retention Usually, concentration polarization increases
Retention, also sometimes called rejection, is a retention of lower-molecular weight species.
function of molecular size and shape. Nominal A membrane with a 100K NMWL may reject
cut-off levels, defined with model solutes, are 1020% of albumin in a 0.1% solution of pure
convenient indicators. Degree of hydration, counter albumin. However, in the presence of larger solutes
ions, and steric effects can cause molecules with such as IgG, it may reject 90% of the albumin.
similar molecular weights to exhibit very different Concentration polarization makes it very difficult
retention behavior. Many biological macromolecules 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
tend to aggregate, or change conformation under to be separated differ in size by at least an order
varying conditions of pH and ionic strength, so that of magnitude.
effective size may be much larger than the native
molecule, causing increased rejection. Solute/
solvent and solute/solute interactions in the sample
Figure 3. High concentrations
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
Ultrafiltration separates proteins from soluble salts.
recommend a smaller differential between the size Concentration polarization slows down filtration.
The proteins form a gel layer on the membrane surface.
7
Flux (UF Flow Rate) Many antifoams exhibit a phenomenon called
cloud point. As temperatures increase, antifoam
During ultrafiltration, it is important to balance speed
comes out of solution, forming a second phase.
with retention to obtain optimal performance. A
Increasing temperature above the cloud point
membranes flux is defined as the flow rate divided
causes flux to decrease.
by the membrane area. Using membranes with
higher NMWL ratings will increase the flow, but pH
at the same time lower the retention. A membrane Changing solution pH often changes molecular
should be selected for required rejection, consistent structure. This is especially true for proteins. At its
with desired flow rate. This is determined by surface isoelectric point, a protein begins to precipitate,
area, macrosolute type, solubility, concentration causing a flux decrease.
and diffusivity, membrane type, temperature effects
on viscosity and, to some extent, pressure. When Fouling
concentration polarization is rate-controlling, flux Flux decrease due to concentration polarization
is affected by solute concentration, fluid velocity, should not be confused with the effect of membrane
flow channel dimensions, and temperature. fouling. Fouling is usually the deposition and
accumulation of submicron particles and solute on
Effects of Operating Parameters on Flux the membrane surface and/or crystallization and
precipitation of smaller solutes on or within the
Pressure
pores of the membrane. There may be a chemical
When ultrafiltering dilute protein solutions or colloid
interaction with the membrane.
suspensions, flux will increase with increasing
transmembrane pressure (TMP). These effects are Importance of Recovery
most apparent when operating under controlled
While rejection is used to characterize membrane
positive pressure, such as when using a stirred
performance, it does not always directly correlate
cell. When the process is membrane-controlled
with solute recovery from a sample or volume.
(i.e., when the resistance of the gel layer is much
Actual solute recoverythe amount of material
smaller than that of the membrane), the flux-pressure
recovered after ultrafiltrationis generally based
relationship is linear. When the process is controlled
on mass balance calculations.
by polarization (e.g., when the resistance of the
In many cases, especially when working with
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
8
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 the fluid flow occurs in the direction normal to the membrane surface,
membrane under an applied pressure. Particulates that are too large to so NFF is a more descriptive and preferred name. NFF can be used
pass through the pores of the membrane accumulate at the membrane for sterile filtration of clean streams, clarifying prefiltration, and virus/
surface or in the depth of the filtration media, while smaller molecules protein separations.
pass through to the downstream side. This type of process is often In tangential flow filtration (TFF), the fluid is pumped tangentially
called dead-end filtration. However, the term normal indicates that along the surface of the membrane. An applied pressure serves to
force a portion of the fluid through the membrane to the filtrate side.
Normal Flow Filtration Tangential Flow Filtration As in NFF, particulates and macromolecules that are too large to
pass through the membrane pores are retained on the upstream side.
Feed Flow Pressure Pressure
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 With very small sample volumes, dilution of the
of separating macromolecular mixtures into size- sample before the initial concentration spin can
graded classes either by direct ultrafiltration or by often decrease salt concentration to an acceptable
diafiltration. Diafiltration removes microsolutes by level. For example, if a 200 L sample containing
adding solvent to the solution being ultrafiltered 100 mM salt is diluted to 4,000 L before concen-
at a rate equal to the UF rate, independent of tration in an Amicon Ultra centrifugal filter unit, the
microspecies concentration. This rapid, efficient salt concentration in the 4,000 L sample will be
process washes microspecies from the solution at 5 mM. The concentrate will still contain 5 mM
constant volume, thereby purifying the retained salt. If more complete salt removal is desired, a
species. This process is most effective if the passing re-dilution/spin cycle should be added. In this
molecules are at least 10 times smaller than the example, if the original spin ended with 50 L of
molecules to be retained and concentrated by the retentate, redilution to 4,000 L results in 0.06 mM
membrane. Diafiltration is useful for sample desalt- salt concentration. The sample can then be recon-
ing and buffer exchange. centrated to 50 L in an Amicon Ultra centrifugal
When diafiltration is used for sample desalting filter device.
or buffer exchange, there is no resulting change in Diafiltration can be a continuous or a discontinu-
buffer composition. A solution volume with 100 mM ous process. In continuous diafiltration, such
salt still contains 100 mM salt after the initial as in a stirred cell or a TFF device, the solution is
concentration spin. Rediluting the retentate with water maintained at a fixed volume while solvent flows
and spinning again effectively decreases the salt continuously through the mixture. Salts and other
concentration of the sample by the concentration microsolutes are steadily removed by convective
factor of the ultrafiltration. For example, if a 4,000 L transport. Microsolute exchange can be accom-
sample containing 100 mM salt is concentrated to plished using the same principle. Constant operator
50 L (80X) in an Amicon Ultra centrifugal filter unit, attention is not required and the possibility of solute
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
100 mM 100 mM 10 mM 10 mM
NaCl NaCl NaCl NaCl
10
Dialysis vs. Diafiltration Table 1. Comparison of diafiltration and dialysis
Dialysis is a traditional method for removing Diafiltration Dialysis
microsolutes or exchanging solvents. It is a slow Transport convective with solvent, Transport diffusion-controlled,
diffusive process generally employing regenerated- independent of microsolute dependent on type of microsolute.
cellulose tubing as the barrier membrane. In dialysis, composition.
the process solution and exchange solvent are Rapid rate. Fractional removal Slow transport. Lower efficiency
on opposite sides of the semi-permeable barrier independent of content. with decreased microsolute
membrane through which permeating microsolutes concentration.
diffuse. The permeation rate of solutes from sample Ultrafiltration rate reduced Marked temperature dependence
with decreased temperature (reduced transport at lower
to dialysate is a direct ratio to the solute concentra-
(net effect not as marked). temperature).
tion and inversely proportional to the solutes
At elevated macrosolute content, Microsolute transport relatively
molecular weight. Desalting by dialysis is time-
ultrafiltration rate reduced. unaffected by macrosolute content.
consuming and relatively inefficient at low
Minimal exchange solvent Frequent dialysate change.
concentrations. required; easily contained in Recirculation about bags to
Millipores Amicon centrifugal concentrators reservoir. maximize transport.
provide a fast, convenient, high-recovery alternative Simple automation with endpoint Automation possible with complex
to dialysis or precipitation without diluting samples. control apparatus. equipment.
The relative merits of diafiltration and dialysis are
summarized in the Table 1.
Fractionation
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
Fractionation is the process of separating a mixture Factors to consider for efficient fractionation:
into its components using a combination of physico- 1. Selection of the appropriate molecular weight
chemical properties of the solute. Ultrafiltration cutoff: Selecting the appropriate MWCO of the
membranes have been used for fractionation of membrane is critical to ensure efficient fraction-
protein solutions on the basis of size1. This tech- ation. The size of the retained and the passing
nique is also called membrane partitioning chroma- species affect the selection criteria for the
tography1,2. Here, proteins larger than the pore size membrane pore size5.
are retained and those smaller than the pore size
pass through into the permeate. Since fractionation 1. Molecular weight of retained MWCO x 0.9
is rarely absolute, acceptability of the results will 2. MWCO Molecular weight of passing x 3
depend on whether the passing solute, retained
3. Molecular weight of retained
solute or both are of interest to the researcher and Molecular weight of passing x 3
their levels of purity and yield. Hence fractionation
efficiency is defined as a function of yield and purity Example separation: Separating a 10 kDa
relative to the starting solution3. 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 i.e. 87.5%. This relation holds true for the most
24 kDa using a 30K MWCO, fractionation will part since the total amount of the retained solute
not be successful because the 24 kDa species is is unchanged. With multiple spins, more of the
only 80% of the pore size of the 30K MWCO passing solute goes through into the permeate,
and it may leak into the permeate. Thus, careful leaving the retained fraction purer and increasing
attention needs to be paid to the size of the the yield of the passing solute.
solutes and the available MWCOs. 4. A serial fractionation strategy for compartmen-
If the application requires the retained species, talizing unknown or complex mixtures:
then the rules are reversed. For maximal purity, Our recommendation for fractionation of
the MWCO chosen should be very open, and for unknown mixtures is to start by separating the
maximal yield, the MWCO should be very tight. proteins using the highest MWCO available.
2. Starting concentration of proteins: Starting The permeate fraction from this separation is
concentrations of protein solutions affect fractionated on the next highest MWCO and
fractionation efficiency4,5. When fractionating so on, serially. Thus, the permeate fraction from
with an Amicon Ultra device, the starting a device with a 100K MWCO membrane
concentration of the passing solute, to 10 mg/ contains proteins <100 kDa and has undetect-
mL does not affect yields or purity of the solute in able amounts of proteins with higher molecular
the permeate fraction. At the other end, for dilute weights. Similarly, the permeate from the 50K
proteins, where the concentrations are below MWCO contains proteins <50 kDa and so on.
0.5 mg/mL, the polarizing gel-like layer does A point to note is that the efficiency of removing
not foul the membrane. In this case we recom- proteins higher than the cutoff depends on the
mend using the lowest MWCO possible to size difference between the solute and the
prevent any trace amounts of retained solute MWCO, and the starting concentration of that
appearing in the filtrate. solute. Smaller solutes will pass through preferen-
Example separation: Consider the separation of tially compared to the larger ones.*
a 17 kDa protein from a 66 kDa protein. If a
MWCO of 50K is chosen (ratio of MWCO to References
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 1. Blatt WF, Hudson BG, Robinson SM, Zipilivan
solute are not affected even when the starting EM. Fractionation of protein solutions by
concentration of the retained is greater than membrane partition chromatography.
5 mg/mL. However, if the 30K MWCO is Nature 1967;(216)511-513.
chosen to maximize purity of the passing solute, 2. Blatt WF. Membrane partition chromatography:
then yields are significantly affected as concen- a tool for fractionation of protein mixtures.
tration of the retained increases to 5 mg/mL and J Agric Food Chem 1971;19:589-594.
beyond. This is due to the fact that the ratio of
3. Ngiam SH, Bracewell DG, Zhou Y, Titchener-
the MWCO to the passing solute is ~1.8X.
Hooker NJ. Quantifying process tradeoffs in
3. Multiple diafiltration steps to increase yields: the operation of chromatographic sequences.
In order to increase yields of the passing solute Biotechnol Prog 2003;19:1315-1322.
and to increase purity of the retained solute,
4. Robertson BC, Zydney AL. Polarization and
multiple spins are necessary. This approach is
adsorption effects on sieving in membrane
also called as diafiltration, where the retentate
protein filtration. ASAIO Trans 1987;33:
fraction is diluted back to the starting volume and
118-122.
the sample is centrifuged again. For e.g., if 50%
5. Nel RG, Oppenheim SF, Rodgers VG. Effects of
of the passing solute was recovered in the filtrate
solution properties on solute and permeate flux
in the first spin, 50% of the remaining, i.e., 25%
in bovine serum albumin-IgG ultrafiltration.
would be recovered in a subsequent spin. The
Biotechnol Prog 1994;10:539-542.
total for 3 spins would be 50 + 25 + 12.5,
12
Membranes and Devices Membrane Selection . . . . . . . . . . . . . . . . . . . . 14
Device Selection . . . . . . . . . . . . . . . . . . . . . . . 16
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 Biomax Ultrafiltration Membrane
devices used for sample concentration, purification, To concentrate or desalt higher volumes of more
and desalting or buffer exchange of soluble concentrated samples (recommended for protein
macromolecules. Millipore products are also concentrations greater than 1.0 mg/mL), use
available for applications such as cleaning up Biomax polyethersulfone (PES) ultrafiltration mem-
PCR reactions, separating protein-bound from free branes. Biomax membranes are recommended for
ligands, removing restriction enzymes, and recover- samples such as serum, plasma, or conditioned
ing oligonucleotides from agarose gels. This section tissue culture media.
provides information to aid in choosing the correct
product for a particular application. Durapore Microporous Membrane
Millipore offers three distinct types of membranes To clarify biological samples, recover DNA from
to choose from. This section will describe these agarose gels, retain chromatography resins or
three types of membranes and then provide suspended solid media, use Durapore hydrophilic
information about choosing the correct membrane PVDF microporous membranes. Durapore mem-
based on typical recoveries or application. branes allow all soluble protein and nucleic acids
to pass, retaining sub-cellular fragments, whole cell
Types of Membranes and particulate materials. Durapore membranes are
extremely hydrophilic, and they provide the lowest
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
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.
14
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)
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.
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%
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
17
Amicon Ultra-4 Centrifugal Filters
1 4 m L
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)
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
Recover
Purified
Sample
19
Amicon Ultra-15 Centrifugal Filters
mL
15
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)
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
21
Centriprep Centrifugal Filters
5 15 m L
Protocol
Ordering Information
Description Membrane NMWL Qty/Pk* Catalogue No.
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
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
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
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
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
Ordering Information
Collect
Particulates
Sample
Filtrate
25
Pellicon XL Cassettes and
12 L
125
Process Flux (Lmh)
100
75
50
25
0
0 1 10 100
Protein Concentration (g/L)
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
27
Pellicon 2 Mini-Cassettes
L
10
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
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
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.
Model 8003 Model 8010 Model 8050 Model 8200 Model 8400
b r adn eC ePl ro
Ordering Information
s arnd
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
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
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
Affinity Purification . . . . . . . . . . . . . . . . . . . . . 46
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 Method
Amicon centrifugal devices from Millipore are ideal 1. Select the device with the appropriate NMWL
for removal or exchange of salts, sugars, nucleo- and volume for the application.
tides, and non-aqueous solvents, as well as other 2. Add the sample to the reservoir of the
materials of low molecular weight. They also serve centrifugal device.
to separate free from bound species.
3. If the sample is smaller than the maximum
Millipore centrifugal concentrators provide fast,
volume, it can be diluted up to the maximum
convenient, high-recovery alternatives to dialysis
volume before the first centrifugation step.
and ethanol precipitation. Sample dilution, often
This will help increase the salt removal.
associated with spin columns, is not a problem.
4. Centrifuge at the specified g-force for the
Salt transfer across the membrane is efficient and
recommended amount of time.
independent of microsolute concentration or size.
5. Remove the initial filtrate from the filtrate tube
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
34
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.
35
Recombinant IDO was expressed in E. coli BL21 developed for PKR, and PKR has been purified using
(DE3) cells utilizing the pET 28a (+) vector system. three chromatography steps on Agarose-Heparin,
In this system, a hexahistidyl tag was fused to full- Agarose-Poly (I), Poly (C) and Sephacryl S-200 gel
size IDO at the N-terminus with a spacer sequence filtration columns. After the last step, PKR-containing
and a thrombin cleavage site. The protein was column fractions were pooled and concentrated
purified by conventional His-tag purification using Amicon Ultra-15 30K NMWL devices. The
methods and eluted with imidazole. The histidine concentration step was necessary for long-term
tag was removed by thrombin cleavage. Final protein storage. Table 3 shows the protein recovery
purification was done by gel filtration chromatogra- results obtained after four concentrations. Over 90%
phy G-75. Amicon Ultra-15 centrifugal devices recovery was obtained and no protein loss to the
were used to concentrate the IDO fractions from filtrate was observed.
an initial concentration of ~0.5 mg/mL to a Amicon Ultra-15 30K NMWL devices were also
final concentration of 10 mg/mL. Samples were used for exchanging buffer for PKR autophosphory-
analyzed by SDS-PAGE using a 12.5% polyacryl- lation (activation) assay. 200 L of 6.301 mg/mL
amide gel (Figure 1). In addition, it was shown that PKR in Protein Storage buffer (20 mM HEPES,
no IDO activity loss was observed after concentra- 1 M NaCl, 10 mM b-mercaptoethanol, 0.1 mM
tion using an Amicon Ultra device. EDTA, 10% glycerol, pH 7.5) was diluted to 15 mL
with Phosphorylation Buffer (20 mM HEPES,
Concentration of PKR and Buffer Exchange 50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 1 mM
Courtesy of Peter A. Lemaire and DTT, pH 7.5) and re-concentrated three times using
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
36
Amicon Ultra devices at 3000 x g for 20 minutes at
4 C. The filtrates from the three steps were pooled Figure 2.
and the total amount of protein in all samples was 100
determined by UV absorption A280.
80
References
1. Samuel CE. Antiviral actions of interferon. 8. Cuddihy AR, et al. Double-stranded-RNA-
Interferon-regulated cellular proteins and activated protein kinase PKR enhances tran-
their surprisingly selective antiviral activities. 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
Virology 1991;183(1):111. Mol Cell Biol 1999;19(4):247584.
2. Hovanessian AG. The double stranded RNA- 9. Demarchi F, Gutierrez MI, Giacca M. Human
activated protein kinase induced by interferon: immunodeficiency virus type 1 Tat protein
dsRNA-PK. J Interferon Res 1989;9(6):641 7. activates transcription factor NF-kappaB
3. Lebleu B, et al. Interferon, double-stranded through the cellular interferon-inducible, double-
RNA, and protein phosphorylation. Proc Natl stranded RNA-dependent protein kinase, PKR.
Acad Sci USA 1976;73(9):310711. J Virol 1999; 73(8):70806.
4. Samuel CE. Mechanism of interferon action: 10. Petryshyn R, et al. Effect of interferon on protein
phosphorylation of protein synthesis initiation translation during growth stages of 3T3 cells.
factor eIF-2 in interferon-treated human cells by Arch Biochem Biophys 1996;326(2):2907.
a ribosome-associated kinase processing site 11. Barber GN. Host defense, viruses and apopto-
specificity similar to hemin-regulated rabbit sis. Cell Death Differ 2001;8(2):11326.
reticulocyte kinase. Proc Natl Acad Sci USA 12. Tan SL, Katze MG. The emerging role of the
1979;76(2):6004. interferon-induced PKR protein kinase as a
5. Koromilas AE, et al. Malignant transformation apoptotic effector: A new face of death?
by a mutant of the IFN-inducible dsRNA- J Interferon Cytokine Res 1999;19:54354.
dependent protein kinase. Science 1992; 13. Balachandran S, et al. Activation of the
257:16859. dsRNA-dependent protein kinase, PKR, induces
6. Meurs E, et al. Tumor supressor function of apoptosis through FADD-mediated death
interferon-induced double-stranded RNA signaling. EMBO J, 1998;17(23):6888902.
activated protein kinase. Proc Natl Acad 14. Osman F, et al. A cis-acting element in the 3-
Sci USA 1993;90:2326. untranslated region of human TNF-alpha mRNA
7. Wong AH, et al. Physical association between renders splicing dependent on the activation of
STAT1 and the interferon-inducible protein protein kinase PKR. Genes Dev 1999;13(24):
kinase PKR and implications for interferon and 328093.
double-stranded RNA signaling pathways.
EMBO J, 1997;16(6):1291304.
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
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 Usually protein concentration is achieved by
most commonly used methods in proteome analysis. protein precipitation with acetone or TCA. The
Briefly, proteins are separated by their isoelectric disadvantage of protein precipitation is that some
point (first dimension separation) followed by SDS- of the proteins become insoluble and can not be
PAGE separation by molecular weight (second resolubilized in IPG buffer. Another disadvantage is
dimension separation). Although it is a powerful that many salts become insoluble in acetone and
method for simultaneously displaying hundreds precipitate along with the proteins.
of proteins, 2DE presents a challenge for sample Ultrafiltration (UF) can achieve protein concentra-
preparation. Salts and ionic detergents are common tion and desalting in one step. Figures 1 and 2
chemicals contaminating biological samples that present two examples of protein preparation for
are not compatible with 2DE separation; however 2DE by acetone precipitation and ultrafiltration.
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
they are often required to solubilize proteins from Both examples demonstrate that UF provides more
cells and tissues. The high concentration of salts efficient salt removal and allows better separations
combined with the relatively low protein content and improved resolution of protein spots in
make samples completely unsuitable for isoelectric two-dimensional electrophoresis.
focusing.
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 Methods
A biomarker can be defined as a molecule that Preparation of Serum Peptides by UF and SPE
indicates an alteration in physiology. Biomarkers One milliliter of human serum, with or without
play an essential role in the drug discovery and acetonitrile, was filtered using Amicon Ultra-4
development process. They provide powerful clues 10K NMWL centrifugal devices. The devices were
to genetic susceptibility, disease progression, and centrifuged in a swinging bucket rotor for 15 to
predisposition, as well as drug response and the 30 minutes at 3000 x g. Ten microliters of the
physiological and metabolic profiles of diseases filtrate was acidified with 5 L of 1% TFA, concen-
and drug responses. Biomarkers can also provide trated with ZipTipC18 pipette tips. Co-elution was
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
Figure 1.
A B C
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 References
30K NMWL device. We observed higher quality
1. Ardekani AM, Liotta LA, Petricoin III. Expert Rev
spectra and an increased number of MALDI-TOF
Mol Diagn 2002;2:12.
detected peptides if the serum was ultrafiltered.
Further improvement of spectra can be achieved 2. Carter D, Douglass JF, Cornellison CD, Retter
by reverse phase chromatography, which concen- MW, Johnson JC, Bennington AA, Fleming TP,
trates and desalts the peptides. The ZipTipC18 Reed SG, Houghton RL, Diamond TS, Vedvick
pipette tip is a convenient and efficient tool for TS. Biochemistry 2002;41:6714.
micro-scale sample preparation prior to mass 3. Wellmann A, Wollscheid V, Lu H, Ma ZL,
spectrometry. Figure 2 shows the MALDI-TOF Albers P, Schutze K, Rohde V, Behrens P,
spectra of rat serum peptides prepared with Dreschers S, Ko Y, Wernert N. Int J Mol Med
ZipTipC18 pipette tip out of (A) straight serum, 2002;9:341.
(B) 50% acetonitrile supernatant and (C) serum 4. Petricoin EF, Ardekani AM, Hitt BA, Levine PJ,
processed by ultrafiltration. The last method Fusaro VA, Steiberg SM, Mills GB, Simone C,
provided stronger MALDI-TOF signal, higher Fishman DA, Kohn EC, Liotta LA. Lancet 2002;
signal-to-noise ratio and double the number of 359:572.
detected peptides. 5. Bischoff R, Luider TM. J Chrom B 2004;803:
The addition of 5 to 10 % acetonitrile to serum 2740.
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 6. Stevens EV, Liotta LA, Kohn EC. J Gynecol
ultrafiltration was shown to improve the detection Cancer 2003;13:1339.
of serum peptides, the MALDI spectrum and the
7. Schulz-Knappe P, Schrader M, Standker L,
overall signal intensity.
Richter R, Hess R, Jurgens M, Forssmann W-G.
J Chromatogr A 1997;776:125132.
Conclusion
8. Basso D, Valerio A, Seraglia R, Mazzza S,
For the analysis of serum peptides, complexity
Piva MG, Greco E, Fogar P, Gall N,
reduction by eliminating higher molecular weight
Pedrazzoli S, Tiengo A, and Plebani M.
proteins is critical for high resolution mass spec-
Pancreas 2002;24:814.
trometry. Efficient separation of peptides from the
9. Prazeres S, Santos MA, Ferreira HG, Sobrinho
majority of proteins and salts can be achieved by
LG. Clin Endocrinol (Oxf) 2003;58:68690.
sample ultrafiltration. We have shown the effective
use of Amicon Ultra-4 30K NMWL centrifugal 10. Tirumalai RS, Chan KC, Prieto DA, Issaq HJ,
devices for the preparation of peptides. Other Conrads TP, Veenstra TD, Mol Cell Proteomics
molecular weight cut-offs can be utilized depending 2003;10:1096103.
on the desired range of peptides. The method can 11. Chernokalskaya E, Gutierrez S, Pitt AM,
be used directly in combination with ZipTipC18 Leonard J. Electrophoresis 2004;25:
pipette tips for peptide identification by MS/MS 24612468.
or as a first step prior to further surface-mediated 12. Millipore Case Study PR0001EN00.
enrichment using SELDI-TOF methods. These sample Automating Multiplexed Cytokine Assays
preparation protocols may also be applicable to manuscript in preparation.
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
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
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
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
46
Materials Method for IgG Purification
Ultrafree-MC 0.45 m centrifugal devices Solutions
(Millipore cat. no. UFC3 0HV 00) PROSEP-A binding buffer A: 1.5 M Glycine/
PROSEP-A high capacity resin NaOH, 3 M NaCl, pH 9.0
(Millipore cat. no. 1131 118 26) PROSEP-A elution buffer B2: 0.2 M Glycine/
Rabbit serum Gibco (Invitrogen lot no. HCl, pH 2.5
1132782) PROSEP-A neutralization buffer: 1 M Tris/HCl,
Micro-centrifuge Biofuge Pico pH 9.0
(Heraeus instruments)
Procedure
Jouan CR1822 fixed angle rotor centrifuge
1. 200 mg of PROSEP-A media were placed in
Xcell SureLock Mini-cell vertical electrophoresis Ultrafree-MC 0.45 m filter basket.
system (Invitrogen cat. no. EI0001)
2. The columns were equilibrated with 400 L of
NuPage NOVEX Bis-Tris 4 12%, 1 mm thick, binding buffer A and centrifuged for 1 minute
15 well SDS gels, (Invitrogen cat. no. NP0323) at 100 x g.
NuPage Sample Reducing agent (10X) 3. 200 L of rabbit serum were diluted 1:1 with
(Invitrogen cat. no. NP009) binding buffer and the entire volume was loaded
NuPage SDS Sample Buffer (4X) into the spin column containing PROSEP-A resin.
(Invitrogen cat. no. NP007) 4. Devices were placed on a shaker for 15 minutes
at room temperature and centrifuged at 100 x g
SimplyBlue SafeStain Coomassie G-250 stain
for 5 minutes. Flow-through was collected for
(Invitrogen cat. no. LC6060)
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 5. 500 L of the clarified lysate were added to
the resin.
Solutions
6. The His-tagged proteins were bound for
Lysis buffer: 50 mM sodium phosphate, 300 mM 30 minutes with light agitation.
sodium chloride, 10 mM immidazole, pH 7 7. The lysate was removed by centrifugation at
Binding buffer: 50 mM sodium phosphate, 500 x g for 10 minutes.
300 mM sodium chloride, 10 mM immidazole, 8. The resin was washed with 500 L of wash
pH 7 buffer for 5 minutes with agitation. The wash
Wash buffer: 50 mM sodium phosphate, 300 mM solution was removed by centrifugation for
sodium chloride, 20 mM immidazole, pH 7 5 minutes at 500 x g. This step was repeated
two more times.
Elution buffer: 50 mM sodium phosphate,
300 mM sodium chloride, 250 mM immidazole, 9. 250 L of elution buffer were added to the
pH 7 Ultrafree-MC device and mixed for 5 minutes.
Purified protein was recovered by centrifugation
1 mg/mL Lysozyme stock
at 500 x g for 1 minute.
Benzonase
Results
Procedure
The results of purifying rabbit IgG using Ultrafree-
1. Recombinant proteins were expressed in
MC centrifugal devices are shown in Figure 1.
Escherichia coli.
The device was challenged with approximately
2. Cells were prepared at a 10X concentration 14 mg of total protein, with an estimated IgG
using lysis buffer. Lysozyme was added to a content of 1.52 mg. The original serum, flow-
concentration of 0.1 mg/mL. To reduce the through, three washes and two eluted fractions
viscosity, benzonase was added to the lysate. were analyzed by SDS-PAGE. The total amount of
The lysates were clarified by centrifugation. purified IgG, as estimated by OD280, was 1.2 mg
3. 200 L of the 50% resin slurry were added to and 1.1 mg on two devices processed in parallel.
the Ultrafree-MC device and the residual fluid The whole procedure was completed in less than
was removed by centrifugation for 1 minute at one hour. This method can be useful for monitoring
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
48
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 Results
Centrifugal devices containing ultrafiltration mem- Figure 1 shows the SDS-PAGE gel of FITC-labeled
branes are ideal for the removal or exchange of BSA before and after each of four diafiltration cycles.
salts, sugars, nucleotides, non-aqueous solvents, Unincorporated FITC is clearly visible in the starting
and other materials of low molecular weight. They material and in the first filtrate. After only one cycle
also serve to separate free from bound species. of ultrafiltration, the majority of the free label is
For the removal of unincorporated label in protein removed. Subsequent cycles of filtration result in
labeling applications, Millipore centrifugal devices BSA that is virtually free of unincorporated FITC.
provide fast, convenient, high-recovery alternatives Fluorescence measurement offers a more sensitive
to gel filtration. Sample dilution, often associated method to monitor the ultrafiltration process. Figure
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
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
(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
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
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
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
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
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.
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 Materials
The measurement of specific proteins in urine is Amicon Ultra-4 device, 4 mL, 10K NMWL
important for the diagnosis and management of
Centrifuge with fixed-angle or swinging bucket
disease states. In most cases, the content of these
rotor capable of 3400 x g
proteins in urine is too low to be detected and
needs to be concentrated. Amicon Ultra-4 devices Kit for microprotein determination
can be used to concentrate urine samples prior to (i.e. Sigma cat. no. 610-A/Brilliant blue G/
clinical laboratory analyses. For example, patients Coomassie blue)
with multiple myeloma exhibit a proliferation of one Pipetter with 200 L tip
antibody-producing plasma cell, which leads to
Electrophoresis (agarose gel) and immunofixation
excess production of free immunoglobulin light
equipment with apparatus and reagents
chains known as Bence-Jones proteins. After sample
enrichment in the Amicon Ultra-4 10K NMWL Method
device, immunofixation electrophoresis can be used
1. Determine the total protein in a 24-hour urine
to identify free light chains (Bence-Jones proteins)
specimen.
in urine by forming a light chain-antibody complex.
Also, agarose electrophoresis can be used to 2. Fill Amicon Ultra-4 device with 4 mL of urine.
quantitate light chains and identify additional low 3. Centrifuge at 3400 x g for 3045 minutes
molecular weight proteins such as albumin, a-1 (approximately 2550 L concentrate volume).
globulins, transferrin and IgG that can be present This produces up to a 160-fold increase in
in renal tubular disorders. Ultrafiltration of urine concentration.
samples in Amicon Ultra-4 devices provides 4. Insert a pipetter into the bottom of the filter unit
reproducible, high sample recovery for electropho- and withdraw the concentrated sample.
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. 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.
56
Acknowledgements Additional Notes
Research using Amicon Ultra devices for urine 1. Amicon Ultra devices can also be used to
concentration in this protocol was conducted by concentrate serum, plasma and cerebrospinal
Mark Merchant, Ph.D. at Helena Laboratories, fluid for similar analyses. A concentration of
Beaumont, TX. approximately 20 mg/mL is required in order to
detect free light chains from diseased patients by
References agarose electrophoresis. Detection by immuno-
1. Tietz, N. Clinical Guide to Laboratory Tests, fixation electrophoresis is 10 times more sensitive
2nd ed. Philadelphia: Saunders, WB; than by agarose electrophoresis.
1990;362363. 2. Normal heterogeneous immunoglobulins may
2. Kahns L. Clinical Chemistry 1991;37: also be seen in urine concentrate with immuno-
15571558. fixation electrophoresis. This ladder effect is
3. Cleveland Clinic homepage. Accessed July comprised of microheterogenous light chains.
2002. www.clevelandclinic.org/myeloma/ Bence-Jones proteins may be within this ladder.
DiagnosisAndTreatmentOf MultipleMyeloma.html To verify the presence of Bence-Jones proteins
requires additional analysis by two-dimensional
4. Christenson RH, et al. Clinical Chemistry
electrophoresis.
1983;29(6):10281030.
3. If there is excess antigen, dilution of the concen-
5. Christenson RH, and Russell ME. Clinical
trate will be required until equilibrium is achieved
Chemistry 1985;31(6):973.
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 Abstract
Stirred cell devices have been successfully used to This study aims to achieve purification and concen-
purify proteins from large volumes of solution for tration of a fusion protein composed of the alpha
many years. However, the need for assembly and and gamma subunits of the human high affinity
cleaning between uses, and the lengthy separation IgE receptor. The alpha-gamma fusion protein is
times, can present difficulties for active, or under- purified and then coupled to Sepharose (GE)
staffed, laboratories. beads to produce an affinity column for isolating
Millipores large volume centrifugal filter devices human IgE antibodies.
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
140
Results
120
Spectrophotometric evaluation of alpha-gamma
Absorbance Units
20
Acknowledgement
0
0 5 10 15 20 25 30
Protocol courtesy of GKT School of Biomedical
Time (min) Sciences, London.
58
Protocols for
Purification of DNA Sequencing Reactions . . . . 60
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 Method
DNA sequencing reactions performed using ABIs DNA was isolated according to standard protocols
BigDye terminator chemistries require the removal and amplified for sequencing analysis on an ABI
of unincorporated dye-terminators and excess salt model 3700 DNA sequencer using polymerase
prior to injection into a sequencer. The traditional reagents and ABIs BigDye Terminator v. 3.1.
method for purifying DNA sequencing reactions is Sample was purified using two experimental
ethanol precipitationa labor intensive method protocols. Results were assessed using electrophero-
requiring multiple spins, incubation, and drying that grams, Phred 20 read lengths, and estimation of
can take up to an hour. Care must also be taken to dye blobs.
retain the resulting pellet.
Microcon 100 protocol
Microcon 100 devices can remove contaminat-
ing salts and unincorporated dye terminators from 1. Sequence Product (10 L) + 100 L of Montage
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 injection solution in a Microcon 100 device
and read lengths equal to or better than ethanol 2. Spin for 2.5 minutes at 12,000 x g
precipitation in 10 minutes. As capillary-based 3. Add 100 L of injection solution to device
DNA sequencers become more prevalent, the need 4. Spin for 2.5 minutes at 12,000 x g
for single sample purification of SEQ reactions also
5. Resuspend in 20 L of injection solution then
increases.
incubate for 2 minutes
The first protocol achieved a time savings of
6. Invert spin for 2 minutes at 1,000 x g
50 minutes over the traditional ethanol precipitation
method while a second protocol, which relies on 7. Inject into sequence analyzer
less vigorous centrifugation, took approximately The above steps were completed in 10 minutes
as long as ethanol precipitation but excelled in at least one third the time required for ethanol
experiments in which it was desirable to read precipitation.
close to the primer.
Results
Equipment Key parameters for DNA sequence reactions
Instrument: ABI sequencer cat. no.3700 include quality, read length, sequencing accuracy,
ability to read close to the primer, and the presence
Reagent: ABI BigDye Terminator v3.1
of dye blobs. The first three factors may be assessed
Injection parameters: existing parameters using Phred 20 scoring, which is performed auto-
developed for Montage Injection solution. matically via software. Phred 20 corresponds
Data output: Graphs and electropherograms roughly to a 99% probability that a base call is
that show A) removal of dye-terminators (blobs); accurate. Ability to read close to the primer is
B) Read length (Phred 20 scores); C) Primer 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.
60
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.
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 sition. The salt concentration in a sample concen-
available for concentrating nucleic acids: trated by Microcon centrifugal filter devices will be
repeated extractions with n-butanol the same as in the original sample. For desalting,
the concentrated sample is diluted with water or
adsorption to ion exchange resin,
buffer to its original volume and spun again in a
followed by high salt elution
process called diafiltration. This removes the salt
lyophilization by the concentration factor of the ultrafiltration.
The first method has the disadvantage that For example, if a 500 L sample containing
n-butanol concentrates all solutes, including salt, 100 mM salt is concentrated to 25 L (20X
which tends to co-precipitate with DNA upon concentration factor), 95% of the total salt in the
addition of ethanol. The second method, ion sample will be removed. The salt concentration in
exchange, aside from requiring buffers of various the sample will remain at 100 mM. Rediluting the
ionic strengths, yields DNA in a high salt solution. sample to 500 L will bring the salt concentration
The third method, lyophilization, increases the to 5 mM. Concentrating to 25 L once more will
concentration of buffer components, which can remove 99% of the original total salt. The concen-
result in degradation of nucleic acids. trated sample will now be in 5 mM salt. For more
Ultrafiltration membranes retain DNA or RNA but complete salt removal, an additional redilution and
are permeable by smaller ionic buffer components. spinning cycle will remove 99.9% of the initial salt
Ultrafiltration alone does not change buffer compo- 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
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) Several suppliers offer STR assay kits. Each
membranes are used by forensics laboratories to supplier offers several versions of their kits with
isolate genomic DNA for human identification. The different protocols, modes of operation, and
source materials are typically blood stains and/or detection systems to suit the users needs. Millipores
other bodily fluids obtained from crime scenes, UF-based centrifugal devices are specified
criminal suspects, or human remains. in several of these protocols (AmpFLSTR Profiler,
The isolated genomic DNA is used to identify Applied Biosystems; GenePrint STR and GenePrint
individual persons based on their patterns of Short Fluorescent STR Systems, Promega).
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
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
Figure 2. Effect of MWCO and g-force
To enable the use of DNA fragments generated by
on the recovery of a 137 bp PCR product
the Polymerase Chain Reaction (PCR) in downstream
using Amicon Ultra-4 100K device
applications, unincorporated primers, nucleotides
100
and contaminating salts must to be quantitatively
removed. The application of ultrafiltration to PCR
% PCR Recovery
80
68
spin at 2,000 x g. The purified PCR products were Ultrafiltration-based purification relies on size
collected by pipette and the recovered DNA exclusion to affect the separation of single stranded
quantified by a fluorescent SYBR Green I assay primers from the double stranded PCR fragments.
(Molecular Probes). The final sample volume was As discussed above, the major challenge to
87 15 L. The recoveries shown in Figure 1 optimizing ultrafiltration for PCR purification is
demonstrate the high recovery that can be obtained with small, <1000 bp PCR products. Similarly, the
from PCR reactions using this approach. efficiency of primer removal becomes more
The recovery of PCR fragments purified by challenging as the size of the single stranded primer
ultrafiltration can be challenging when the resulting increases. As shown in Figure 4, the ability of an
DNA fragments are <1000 bp in length. The Amicon Ultra device with 30K MWCO membrane
amount of DNA recovered after purification can to remove primers is significantly diminished as the
be maximized by using an Amicon Ultra device primer length approaches 50 bases. In contrast,
with a lower MWCO membrane (e.g., 50K versus when a 50K MWCO membrane is used, primer
100K MWCO) and by centrifuging the devices at removal remains high and is consistent across the
2,000 x g as shown in Figure 2 for a 137 bp PCR range of PCR primers evaluated. Whereas the use
fragment. Although increasing the g-force will speed of a 100K would be expected to quantitatively
processing time, it will do so at the expense of DNA remove all but the largest of primers, purification
recovery. For larger DNAs, the effect is less of small (e.g., <300 bp) PCR products is suboptimal
dramatic (data not shown). when using a 100k MWCO membrane. Therefore,
The quantitative removal of single-stranded DNA an Amicon Ultra-4 device with a 50K MWCO
primers or oligonucleotides from PCR reactions is membrane provides the best balance of primer
critical for downstream applications. In order to removal and recovery when used for PCR
demonstrate the ability of Amicon Ultra devices to purification.
efficiently remove primers, unpurified PCR products
were spiked with 20 pmoles of a DNA reporter 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
primer that had been labeled with fluorescein on its The data presented herein demonstrate the utility of
5 end. This primer (TCAG)5 was designed such that Amicon Ultra-4 devices for the purification of PCR
it had minimal secondary structure. After purification products. By using devices with a 50K MWCO,
of the PCR products with an Amicon Ultra-4 device, users can expect to routinely obtain both high
the amount of primer remaining in the sample was recoveries and efficient primer removal that will
determined by measuring fluorescence against a facilitate downstream applications. Although the
standard curve. The average primer removal in this analysis performed for this study focused on PCR
experiment was 97.7 1.4%. This level of primer purification, the general principles are applicable
removal has been shown to be sufficient for DNA to purification of other nucleic acid preparations.
sequencing and other important PCR-based
applications.
Figure 4. Effect of MWCO on primer
removal in Amicon Ultra devices
Figure 3. Amicon Ultra device efficiently 100
Removal (% of Challenge)
60
80
40
60
20
40
0
20 20 25 48
Primer Length (Bases)
0
137 301 657 1159 Amicon Ultra, 50k Amicon Ultra, 30k
PCR Fragmentation Size (bp)
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
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
71
RNA Purification and Preparation of
Fluorescent cDNA Probe from Human
mRNA with Microcon Centrifugal Filters
Introduction tion of high quality fluorescently labeled probes
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
Figure 1.
Concentration of RNA transcript. RNA is intact
1 2 3
and recovered with high efficiency.
72
Method 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
Generation and Purification of Fluorescently a Microcon 30K NMWL device column. Next
Labeled Probes add the 60 L of Cy5 probe and the 60 L of
1. To anneal primer, mix 2 g of mRNA or Cy3 probe to the same Microcon device.
50100 g total RNA with 4 g of a regular NOTE: If re-purification of Cy dye flow-through is
or anchored oligo-dT primer in a total volume desired, do not combine probes until Wash 2.
of 15.4 L as shown in Table 1.
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.
73
11. Wash 3: Remove flow-through and add Results
450 L 1X TE, 20 g of Cot1 human DNA
RNA recovery is a function of the initial RNA
(20 g/L, Gibco-BRL), 20 g polyA RNA
concentration and the buffer salt concentration.
(10 g/L, Sigma cat. no. P9403) and
Figure 2 shows RNA transcript recovery in
20 g tRNA (10 g/L, Gibco-BRL cat.
Microcon 100K.
no. 15401-011). Spin 710 minutes at
NMWL unit as a function of RNA concentration.
14,000 x g. Look for concentration of the probe
For all concentrations evaluated, retentate recovery
in the Microcon device. The probe usually has
is above 85%, even at an initial concentration as
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
74
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%
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
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)
75
Purification of In Vitro Synthesized
mRNA with Microcon Centrifugal Filters
Introduction Methods
In vitro transcription reactions employing T3, T7 or RNA Transcription
SP6 phage-encoded RNA polymerases are widely For our studies we chose plasmid pGEM-luc
used to synthesize RNA from recombinant vectors containing the luciferase gene (luc) in the center of
containing appropriate promoters. Production of a multiple cloning cassette of the pGEM-11Zf (-)
large amounts of specific RNA is valuable in the plasmid (Promega). DNA template was linearized
preparation of hybridization probes and in vitro with XhoI, followed by enzyme and salt removal by
translation studies; in the synthesis of ribozymes, diafiltration in Microcon 100K NMWL devices.
rRNA, SRP, antisense RNA and substrates for RNA Linearized template was transcribed, using
splicing; and in RNA-protein interaction studies. MEGAscript kit (Ambion) according to the recom-
Microcon centrifugal filters are well suited for mended protocol. After the reaction was completed
the purification of radiolabeled RNA transcripts1. (3 to 4 hours), template DNA was degraded with
Ultrafiltration can simultaneously and efficiently DNase I and the reaction mix added to a Microcon
remove unincorporated ribonucleotides and salts 30K NMWL device filled with 450 L of water. The
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 device was spun for 20 minutes at 12,000 x g in a
molecules retain their integrity and are recovered temperature-controlled centrifuge at 4 C. Purified,
with high yields. concentrated RNA was recovered by inverted spin.
Purity of a transcript is especially important when For the preparation of capped transcript, cap
it is used in in vitro translation systems. Trace analog m7G (5) ppp (5) G (New England
amounts of ethanol, phenol, salts or excess cap Biolabs, Inc.) was included in the transcription
analog used during the synthesis of capped mRNA reaction and the level of GTP reduced (4:1 ratio
can cause a dramatic decrease in translation of cap analog to GTP). To purify the transcript by
efficiency. phenol/chloroform extraction, the reaction mix
After the transcription reaction is complete, was diluted with water and a one-tenth volume of
template DNA is usually degraded by the addition ammonium acetate stop solution was added. The
of DNase I. The RNA is purified by two phenol/ mixture was extracted once with phenol/chloro-
chloroform extractions followed by ethanol form, followed by chloroform extraction. RNA was
precipitation. Other, less popular methods are gel precipitated with isopropanol and the pellet
purification (used predominantly when separation of resuspended in distilled water. Alternatively, LiCl
full-length transcript from shorter RNAs is important, precipitation solution (one-half volume) was added
e.g., ribonuclease protection assays) or LiCl to the reaction mix, followed by incubation at minus
precipitation. 20 C for 1 hour. RNA was pelleted by centrifuga-
A series of experiments was performed in our tion and dissolved in water. Size and integrity of
laboratory to determine the effectiveness of using the in vitro transcription products were assessed by
Microcon devices to purify in vitro synthesized running an aliquot of the purified RNA transcript on
mRNA and in vitro translation studies. Results a formaldehyde/formamide agarose gel. Ethidium
indicate that ultrafiltration can efficiently remove bromide was added to the RNA before lading on
inhibitory contaminants from mRNA preparations, the gel to stain the RNA sample and keep back-
leading to increased translational efficiencies. ground fluorescence low2.
76
Translation In Vitro References
In vitro translations were performed in the Flexi
1. Krowczynska AM. BioSolutions 1993;2(1):12.
Rabbit Reticulate Lysate System (Promega) according 2. Ogretmen B, Ratajczak H, Kats A, Stark BC.
to standard luciferase RNA translation conditions Biotechniques 1993;14(6):932-5.
with minor modifications (Rnasin Ribonuclease
3. Polayes D. Focus 1991;13(4):1302.
inhibitor was omitted and 35S-methionine added).
4. Dasso MC, Jackson RJ. Nucl Acid Res 1989;
Results of translation were analyzed by determina-
17:3129.
tion of percent incorporation of 35S-methionine and
fold stimulation, compared to controls without RNA.
Minimum acceptable stimulation was 8-fold.
Figure 1.
1 2 3 4
Results Comparison of pGem-luc transcript purification
methods. Transcripts were synthesized in 20 L
Aliquots of RNA transcript purified by different
reactions. After DNase I treatment, RNA was
methods (ultrafiltration, phenol extraction and LiCl purified from the reaction mix. 500 ng of
precipitation) were run on a denaturing agarose/ purified RNA were run on 1% agarose/
formaldehyde gel. Results are shown in Figure 1. formaldehyde gel.
The banding pattern of the 1.7 kb RNA transcripts Lane 1: 0.161.77 kb RNA ladder (Gibco BRL)
is identical regardless of purification method. Similar Lane 2: RNA purified in Microcon 30K NMWL
device
results were obtained in the case of capped
Lane 3: RNA purified by phenol extraction
transcript (results not shown).
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
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
Methods
Figure 1.
Nuc
1 2 3
DNA Ladder
P ro t o c o l s fo rNu
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
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 References
DNA samples of up to 49 kb were concentrated 1. Takagi S, Kimura M, Katsuki M. BioTechniques
repeatedly without any loss of sample integrity. 1993;14(2):21821.
Some loss of integrity was observed with a 125 kb 2. PCR is covered by U.S. patents issued to
sample, although it was not complete and repre- Hoffmann-LaRoche, Inc.
sents a small percentage of the total DNA in the
3. Sheng N, Zhang J, Whitton JL, McKee T.
sample. For large fragments of DNA, centrifugal
BioTechniques 1993;14(5):7814.
ultrafiltration provides a fast and efficient method to
4. Ruano G, Pagliaro EM, Schwartz TR, Lamy K,
concentrate or desalt the sample. It results in high
Messina D, Gaensslen RE, Lee HC.
recovery of intact product.
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 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.
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
Results
Variable speed microcentrifuge
Montage PCR filter units were used to purify 100 L
Montage PCR filter unit PCR reactions according to the specified protocol.
Purified water (such as Milli-Q water) or TE buffer After recovering the DNA fragments (n = 10) using
a reverse spin, the samples were separated by
PCR reaction (aqueous phase)
agarose gel electrophoresis. Recoveries of the
Method various PCR products were determined by densitom-
etry (Figure 1).
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
82
Figure 2.
100%
80%
Percent Recovery
60%
40%
20%
0%
137 bp 301 bp 500 bp 657 bp 1159 bp
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
*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 which was added directly to the filtrate and
collect the condensate at the bottom of the micro- incubated, appeared completely intact (lanes
centrifuge tubes, 1 L of 0.5 M disodium EDTA 1114). Since the standard curve indicated that
and 10 L of 5X loading buffer were added to 0.08 units would be detected if it were present,
stop the reaction. we calculated that at least 99.8% of Bgl I was
The negative control was a portion of DNA removed and/or inactivated. Removal, rather than
master mix. Control digests of pBR322 DNA and inactivation, is the probable mechanism by which
pUC19 DNA were carried out separately. Micropure-EZ devices operate, since in separate
experiments we were unable to detect any enzyme
Results and Discussion (irrespective of activity) in Micropure-EZ filtrates
The Bgl I standard curve made with the Micropure- using very sensitive HPLC methods (data not shown).
EZ-filtered sdH2O was indistinguishable from
the standard curve made with untreated sdH2O. Summary
The plasmid DNA (1 g of pUC19 and 1 g of Enzyme removal and excellent DNA recovery are
pBR322) was cut to completion by 2 units of Bgl I accomplished with Micropure-EZ devices in a single
after one hour at 37 C (Figure 2, lanes 1 and 6). 60-second spin without any pre-wetting, binding,
None of the enzymes shown in Tables 1 or 2 was washing or elution step. Cumbersome phenol/
measurably inhibited by Micropure-EZ-filtered chloroform extraction and the associated hazardous
sdH2O. The standard curves indicated that as little waste accumulation are avoided. An additional line
as 0.08 units of Bgl I could be detected by this of evidence for the suitability of Micropure-EZ
assay (lanes 3 and 8). devices in molecular biology applications is that
The devices removed the 50 units of Bgl I as restriction digest purified with Micropure-EZ devices
evidenced by an absence of detectable activity in alone are readily ligated and cloned into plasmid
the filtrates. In the lanes corresponding to the Bgl I DNA (M.H.A.L., unpublished observations).
challenged Micropure-EZ inserts, some activity was Micropure-EZ devices used alone or with Microcon
observed against the decoy pBR322 DNA, as devices permit rapid and efficient sequential
expected during its brief exposure to Bgl I (particu- enzymatic DNA manipulations.
larly evident in lane 14). However, the pUC19,
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
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) Xba I 50 U
ApaL I 10 U In tests, Micropure-EZ devices did
Bgl II 50 U not remove the indicated number of
units of the listed enzymes. It may be
BsoB I 50 U effective in removing a lower number
Cla I 25 U 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
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
88
Concentration using Amicon Ultra-4 or Ultra-15 6. Set retentate pressure by slowly adjusting
devices with 50 kDa NMWL membrane retentate valve clockwise until retentate
1. Clarify phi-6 supernatant using a Stericup pressure is 10 psi.
filter unit (Millipore cat. no. SCGP U05 RE), 7. Adjust pump speed and retentate valve
a Steriflip filter unit (Millipore cat. no. restriction to achieve desired feed pressure
SCGP 005 25), or a Millex-GP syringe filter of 30 psi and retentate pressure of 10 psi.
(Millipore cat. no. SLGP 033 RB) with low- Note: Do not exceed feed pressure of 60 psi.
binding 0.22 m PES membrane.
8. Filter the solution until the desired concentration
2. Load appropriate volume of clarified phi-6
or volume is obtained.
supernatant (4 or 15 mL) into an Amicon Ultra-4
9. Stop pump.
device (Millipore cat. no. UFC8 050 24) or
an Amicon Ultra-15 device (Millipore cat. no. 10. Disconnect pump outlet tubing from pump
UFC9 050 24) containing 50,000 NMWL outlet port and place in product recovery
regenerated cellulose membrane. collection vessel.
89
For large volume concentration, tangential flow 5. Bachrach U, Friedmann A. Appl Microbiol
filtration (TFF) should be used. Table 1 shows phage 1971;22(4): 706-715.
concentration results using a Pellicon XL50 cassette 6. Sambrook J, Fritsch EF, Maniatis T. Molecular
with the Labscale TFF system. The Pellicon XL50 cloning: a laboratory manual, 2nd ed. Cold
cassette couples Millipores ultrafiltration membranes Spring Harbor Laboratory Press, Cold Spring
with the linearly scalable TFF cassette for processing Harbor, N.Y. 1989
501000 mL volumes. The Ultracel membrane with 7. Bahns JT, Liu CM, Chen L. Protein Science
30 kDa NMWL is recommended. Twenty-fold 2004;13:2578-2587.
concentration of phage solution is usually achieved
8. Gill JJ, et al. Chemother 2006;50(9):
in 3050 minutes.
2912-2918.
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.
90
Concentration of Animal Viruses
Using Ultrafiltration
Introduction and adeno-associated virus (AAV)10. Challenges
associated with the use of viral vectors in clinical
Despite the rapid progress in genomics, the biologi-
trials include production of sufficient quantities of
cal function of the majority of the human genes is
clinical grade material and maintaining biosafety of
still unknown. The identification of gene function is
the viral vector. Production and purification methods
an increasingly relevant issue, especially in the
for viral vectors vary both in terms of methodology
search for new targets for improved human disease
and virus yield and quality.
therapy. Functional genomics approaches, which
Generation and purification of viral vectors can
aim at the identification of genes via phenotypes
be labor intensive, costly and may require special
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;
93
High Throughput Applications
The MultiScreen filter plate with Ultracel-10 Purification of Serum Peptides for
membrane provides a new method for high Biomarkers Research
throughput sample prep. The ultrafiltration-based AN2010EN00
filter plate is designed for use with centrifugation This study is a high throughput version of the
and is compatible with standard microtiter plates, application note on page 42 of the Protocols
instrumentation, and liquid handling equipment. section of this handbook.
All of the publications summarized below
can be supplied by your local Millipore office or Enzymatic Activity Recovery
downloaded from www.millipore.com/ultracel. AN2011EN00
This study demonstrates the use of the MultiScreen
Nucleic Acid Purification and Concentration plate with Ultracel-10 membrane for concentrating
AN1040EN00 alkaline phosphatase without loss of biological
The MultiScreen Filter Plate with Ultracel-10 activity and with high reproducibility across
membrane can be used to purify and concentrate the plate.
single-stranded oligonucleotides and double-
stranded DNA fragments, as well as plasmid
and genomic DNA.
94
Glossary
Asymmetric Membrane1 Fouling Reverse Osmosis1
Membrane constituted of two or more Irreversible decline in membrane flux due to Liquid-phase pressure-driven separation
structural planes of non-identical deposition and accumulation of submicron process in which applied transmembrane
morphologies. particles and solutes on the membrane pressure causes selective movement of
surface. Also, crystallization and precipita- solvent against its osmotic pressure
Batch Process
tion of small solutes on the surface and in difference.
A fixed volume of solution contained in a
the pores of the membrane. Not to be
tank to which the concentrate is returned Tangential Flow Filtration (TFF)
confused with concentration polarization.
during the process. Flow through a membrane device in which
Membrane1 the fluid on the upstream side moves
Composite Membrane1
Structure having lateral dimensions much parallel to the membrane surface.
Membrane having chemically or structurally
greater than its thickness, through which
distinct layers. Transmembrane Pressure (TMP)
mass transfer may occur under a variety
The driving force in ultrafiltration. In a stirred
Concentration Polarization of driving forces.
cell, equivalent to gas pressure. In centrifu-
Accumulation of rejected solute on the
Molecular Weight Cut-off (MWCO) gal devices, it is related to g-force. In a
membrane surface. Depends on interactions
See Nominal Molecular Weight Limit. flowing system, TMP decreases as the
of pressure, viscosity, crossflow (tangential)
stream moves from inlet to outlet. Average
velocity, fluid flow conditions, flow channel Nanofiltration1
TMP = [(Pin + Pout)/2] Ppermeate
conditions and temperatures. Pressure-driven membrane-based separation
process in which particles and dissolved Ultrafiltration1
Crossflow (Tangential Flow)
molecules smaller than about 2 nm are Pressure-driven membrane-based separation
Solution flows across (tangential to) a
rejected. process in which particles and dissolved
membrane surface. Facilitates back diffusion
molecules smaller than 0.1 m and larger
of solute from that surface into the bulk Nucleotide Cut-off (NCO)
than about 2 nm are rejected.
solution, counteracting concentration The number of nucleotides in a DNA
polarization. fragment (single- or double-stranded) at Yield
which 90% of the fragment is retained by Amount of species recovered at the end of
Diafiltration
the membrane. the process as a percentage of the amount
Removal of small components from the
present in the feed solution.
retained species during ultrafiltration by Nominal Molecular Weight Limit (NMWL)
adding water or buffer solution to the The molecular weight at which at least 90%
retentate. See page 7 for further discussion. of a globular solute of that MW is retained References
by the membrane. 1. Terminology for membranes and
Dialysis
Diffusive transport of ions or other small Permeate (Filtrate, Ultrafiltrate) membrane processes (IUPAC
molecules through a membrane barrier The solution passing through the membrane, Recommendations 1996). IUPAC,
that contacts solvent on both sides. containing solvent and solutes not retained Journal
by the membrane. of Membrane Science.1996;120(1):
Downstream1 14959.
Side of a membrane from which the Plugging
permeate emerges. Accumulation of debris on the fluid flow
path, restricting or blocking flow.
Feed (Sample)
The starting solution (sometimes the solution Rejection App e nd i x G l o s s a r y
remaining upstream of the membrane). The fraction of solute held back by the
membrane. Can be measured at any point
Fluid Velocity
in the process or averaged over the run.
The flow rate of solution across the
membrane surface in cross (tangential) flow. Retentate (Reject Stream, Concentrate)
Related to hydraulic pressure drop. The solution containing the retained
(rejected) species.
Flux
The filtration rate through the membrane Retention Factor1 (rF)
per unit area. 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
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
96
Patent
PCR is covered by US patents issued to Hoffmann-LaRoche, Inc.
Trademarks
Millipore, Amicon, Biomax, Centricon, Centriprep, Durapore, Immobilon, Labscale, Microcon,
Micropure, Milli-Q, Minicon, Montage, MultiScreen, Pellicon, Prep/Scale, PROSEP, Steriflip,
Ultracel, Ultrafree, Zip, and ZipTip are trademarks of Millipore Corporation.
AmpFLSTR, BigDye and Voyager-DE are trademarks of Applera Corporation or a subsidiary.
BacPAK and Talon are trademarks of Becton, Dickinson and Company.
Biofuge is a trademark of Kendro Laboratory Products, Gmbh.
CHEF-DR and Chelex are trademarks of Bio-Rad Laboratories.
DryStrips, Sephacryl, Sepharose, and Storm are trademarks of GE Healthcare.
GenePrint, Flexi and Riboprobe are trademarks of Promega Corporation.
Kodak is a trademark of Eastman Kodak Company.
MEGAscript is a trademark of Ambion Inc.
NuPage, SimplyBlue, Superscript and Xcell Surelock are trademarks of Invitrogen Corporation.
ProteoPrep is a trademark of Sigma-Aldrich Biotechnology LP.
ProteomIQ, ElectrophoretIQ, IsoelectrIQ, and GelChips are trademarks of Proteome Systems.
SeaKem is a trademark of FMC.
SpectraFLUOR is a trademark of Tecan Group AG.
SpectraMax is a trademark of Molecular Devices Corporation.
Speed Vac is a trademark of Thermo Savant.
SureBlue is a trademark of Kirkegaard & Perry Laboratories, Inc.
SYBR is a trademark of Molecular Probes, Inc.
Triton is a trademark of Union Carbide Chemicals & Plastics Technology Corporation.
Tween is a trademark of Atlas Powder Company.
Vent is a trademark of New England Biolabs, Inc.
Additional Resources for Life Scientists
Ultrafiltration
applications, including blotting, DNA
and Upstate, which are now part of Millipore.
sequencing sample preparation, sterile
Visit www.millipore.com/immunodetection.
filtration, and MS sample prep. To contact a
Specialist, call your local office or submit a
question at www.millipore.com/techservice.
To access our library of frequently asked
questions, go to www.millipore.com/faqs.
Application and Product Guide