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forensics proteins
Ultrafiltration
Application and Product Guide
www.millipore.com
For over 50 years, Millipore has helped improve laboratory productivity and efficiency for researchers worldwide by delivering innovative products |and services backed by the highest levels of quality and expertise. With the recent acquisitions of Upstate, Chemicon and Linco, we now offer a wide range of antibodies; stem cell-related products; protein immunodetection kits; and drug profiling products and services. The addition of these core products and capabilities to our existing sample preparation and laboratory water offering creates an extensive portfolio of comprehensive solutions for immunodetection, cell biology, and drug discovery.
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Table of Contents
overview of MeMbrane filTraTion
Membrane Processes . . . . . . . . . . . . . . . . . . Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . Mode of Operation . . . . . . . . . . . . . . . . . . . 4 6 9 Removal of Unincorporated Label from Labeled Protein . . . . . . . . . . . . . . . . . . . 50 Rapid Purification of Monoclonal Antibodies . . . 52 A Simple Strategy for Protein Fractionation . . . . 54 Urine Concentration . . . . . . . . . . . . . . . . . . . 56 Use of Centrifugal Filter Devices as an Alternative to Stirred Cells . . . . . . . . . . . . . . . 58
Diafiltration . . . . . . . . . . . . . . . . . . . . . . . . . 10 Fractionation . . . . . . . . . . . . . . . . . . . . . . . . 11
aPPendix
High Throughput Applications . . . . . . . . . . . . 94 Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Ta bl e o f C o n T e n T s
4 6 9
Diafiltration . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Fractionation . . . . . . . . . . . . . . . . . . . . . . . . . 11
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Membrane Processes
Ultrafiltration
Ultrafiltration (UF) is the process of separating extremely small particles and dissolved molecules from fluids. The primary basis for separation is molecular size, although in all filtration applications, the permeability of a filter medium can be affected by the chemical, molecular or electrostatic properties of the sample. Ultrafiltration can only separate molecules which differ by at least an order of magnitude in size. Molecules of similar size can not be separated by ultrafiltration (see Figure 1). Materials ranging in size from 1K to 1000K molecular weight (MW) are retained by certain ultrafiltration membranes, while salts and water will pass through. Colloidal and particulate matter can also be retained. Ultrafiltration membranes can be used both to purify material passing through the filter and also to collect material retained by the filter. Materials significantly smaller than the pore size rating pass through the filter and can be depyrogenated, clarified and separated from high molecular weight contaminants. Materials larger than the pore size rating are retained by the filter and can be concentrated or separated from low molecular weight contaminants. Ultrafiltration is typically used to separate proteins from buffer components for buffer exchange, desalting, or concentration. Ultrafilters are also ideal for removal or exchange of sugars, non-aqueous solvents, the separation of free from protein-bound ligands, the removal of materials of low molecular weight, or the rapid change of ionic and/or pH environment (see Figure 5, page 10). Depending on the protein to be retained, the most frequently used membranes have a nominal molecular weight limit (NMWL) of 3 kDa to 100 kDa. Ultrafiltration is far gentler to solutes than processes such as precipitation. UF is more efficient because it can simultaneously concentrate and desalt solutes. It does not require a phase change, which often denatures labile species, and UF can be performed either at room temperature or in a cold room.
Microfiltration
Microfiltration (MF) is the process of removing particles or biological entities in the 0.025 m to 10.0 m range from fluids by passage through a microporous medium such as a membrane filter. Although micron-sized particles can be removed
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
Reverse Osmosis
0.0001 m 0.001 m
Ultrafiltration
0.01 m 0.1 m
Microfiltration
1 m
Clarification
10 m 100 m
20,000 kDa
B. diminut a
by use of non-membrane or depth materials such as those found in fibrous media, only a membrane filter having a precisely defined pore size can ensure quantitative retention. Membrane filters can be used for final filtration or prefiltration, whereas a depth filter is generally used in clarifying applications where quantitative retention is not required or as a prefilter to prolong the life of a downstream membrane. Membrane and depth filters offer certain advantages and limitations. They can complement each other when used together in a microfiltration process system or fabricated device. The retention boundary defined by a membrane filter can also be used as an analytical tool to validate the integrity and efficiency of a system. For example, in addition to clarifying or sterilizing 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.
Reverse Osmosis
Reverse osmosis (RO) separates salts and small molecules from low molecular weight solutes (typically less than 100 daltons) at relatively high pressures using membranes with NMWLs of 1 kDa or lower. RO membranes are normally rated by their retention of sodium chloride while ultrafiltration membranes are characterized according to the molecular weight of retained solutes. Millipore water purification systems employ both reverse osmosis membranes as well as ultrafiltration membranes. Reverse osmosis systems are primarily used to purify tap water to purities that exceed distilled water quality. Ultrafiltration systems ensure that ultrapure water is free from endotoxins as well as nucleases for critical biological research.
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Recovery
The ultimate aim of ultrafiltration is to maximize recovery of solutes of interest, but there are many membrane characteristics that affect that goal. Factors affecting recovery include: Nominal molecular weight limit (NMWL)/ nucleotide cut-off (NCO) Retention Concentration polarization Flux When using membrane ultrafiltration for sample concentration or desalting, care must be taken to select a membrane (or device) with a NMWL appropriate for the application. Because there are several considerations in determining whether a given solute will or will not be retained by a membrane of a specific cut-off, it is best to choose a device with cut-off at about one half of the molecular weight of the protein to be concentrated. This maximizes protein recovery and minimizes filtration time.
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
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recovery of small PCR products (e.g., ~150 base pairs [bp]) as quickly as possible. For purifications that are driven by vacuum, significantly tighter membranes are typically required to obtain optimal recovery. If the DNA sample is in the presence of high salt (or the device is run at a higher-than-recommended g-force), a significantly reduced DNA recovery may be observed. Under these conditions, higher DNA or RNA recovery can be achieved by using a tighter membrane. However, it will take significantly longer to complete the purification. For applications such as PCR where removal of unincorporated single-stranded primers from double-stranded DNA fragments is required, the molecular weights of the primer and DNA fragment should differ by at least an order of magnitude for efficient separation. Millipore offers devices that are specifically designed for separating and concentrating genomic DNA and PCR products by ultrafiltration.
of the NMWL and the size of the molecule but Millipores recommendation is designed to provide maximum recovery. Please see additional information regarding membrane NMWL selection on page 14.
Concentration Polarization
Another factor affecting the retention characteristics is the potential for membrane fouling, or concentration polarization. This occurs when there is an accumulation of the retained solute on the surface of the membrane. At high concentrations, a gel layer forms that can act as a secondary membrane (Figure 3). This may interfere with passage of the molecules through the membrane and can adversely affect the flow rate. In addition, pH, buffer components, and concentration can result in a protein behaving in an anomalous manner in terms of its retention or passage by UF membranes. During concentration polarization, the gel layer on the membrane surface superimposes its own rejection characteristics on those of the membrane. Usually, concentration polarization increases retention of lower-molecular weight species. A membrane with a 100K NMWL may reject 1020% of albumin in a 0.1% solution of pure albumin. However, in the presence of larger solutes such as IgG, it may reject 90% of the albumin. Concentration polarization makes it very difficult to use UF for solute fractionation unless the solutes to be separated differ in size by at least an order of magnitude.
Retention
Retention, also sometimes called rejection, is a function of molecular size and shape. Nominal cut-off levels, defined with model solutes, are convenient indicators. Degree of hydration, counter ions, and steric effects can cause molecules with similar molecular weights to exhibit very different retention behavior. Many biological macromolecules tend to aggregate, or change conformation under varying conditions of pH and ionic strength, so that effective size may be much larger than the native molecule, causing increased rejection. Solute/ solvent and solute/solute interactions in the sample can also change effective molecular size. For example, some proteins will polymerize under certain concentration and buffer conditions while others (e.g., heme proteins) may break into corresponding subunits. Ionic interactions or stacking can cause small molecules to behave similarly to molecules of greater molecular weight. When this occurs, as in the case of phosphate ions with a 500 NMWL membrane, the small molecules may not effectively permeate the membrane. Millipore recommends the selection of a membrane filter NMWL that is one half the size of the molecule of interest. Other manufacturers may recommend a smaller differential between the size
Ultrafiltration separates proteins from soluble salts. Concentration polarization slows down filtration. The proteins form a gel layer on the membrane surface.
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Many antifoams exhibit a phenomenon called cloud point. As temperatures increase, antifoam comes out of solution, forming a second phase. Increasing temperature above the cloud point causes flux to decrease.
pH
Changing solution pH often changes molecular structure. This is especially true for proteins. At its isoelectric point, a protein begins to precipitate, causing a flux decrease.
Fouling
Flux decrease due to concentration polarization should not be confused with the effect of membrane fouling. Fouling is usually the deposition and accumulation of submicron particles and solute on the membrane surface and/or crystallization and precipitation of smaller solutes on or within the pores of the membrane. There may be a chemical interaction with the membrane.
Importance of Recovery
While rejection is used to characterize membrane performance, it does not always directly correlate with solute recovery from a sample or volume. Actual solute recoverythe amount of material recovered after ultrafiltrationis generally based on mass balance calculations. In many cases, especially when working with small samples of dilute, valuable solutions, the degree of recovery of a target solute is vitally important. In such cases, potential loss by non-specific adsorption must be considered. Different membrane materials adsorb biomolecules to varying degrees. Where maximum recovery is desired, the choice of a membrane with the least non-specific adsorbtivity is essential. Millipores Ultracel regenerated cellulose membranes were specifically developed to minimize non-specific adsorption. Since adsorption is a direct function of membrane and device surface area, device size must be considered when recovery is important. Small, dilute samples should be concentrated with membranes of minimal surface area, commensurate with achievement of reasonable flow rates. Millipore offers a wide range of centrifugal devices, stirred cells, and tangential flow systems with an extensive choice of membrane areas and NMWLs.
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
Concentration
When concentration of the retained species is very low, flux is independent of concentration. As solute concentration rises during operation, increased viscosity and the polarization effect cause flux to decrease.
Temperature
Increasing the operating temperature normally increases UF rates. A higher temperature increases solute diffusivity (typically 33.5% per degree Celsius for proteins) and decreases solution viscosity. Common practice is to operate at the highest temperature tolerated by the solutes and the equipment. An exception to the rule is fermentation broth concentration in the presence of some antifoams.
Mode of Operation
The pressure required for ultrafiltration can be supplied in a number of different ways depending on the product in use. For example, Millipores small volume ultrafiltration products generally use centrifugal force. Pump pressure is used with the tangential-flow filtration (TFF) products and compressed gas is utilized with the stirred cell products. In addition, Millipore provides multiwell ultrafiltration products that utilize vacuum and centrifugation.
Feed Flow
Membrane Filtrate
Membrane Filtrate
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Diafiltration
Millipore membranes provide an inexpensive means of separating macromolecular mixtures into sizegraded classes either by direct ultrafiltration or by diafiltration. Diafiltration removes microsolutes by adding solvent to the solution being ultrafiltered at a rate equal to the UF rate, independent of microspecies concentration. This rapid, efficient process washes microspecies from the solution at constant volume, thereby purifying the retained species. This process is most effective if the passing molecules are at least 10 times smaller than the molecules to be retained and concentrated by the membrane. Diafiltration is useful for sample desalting and buffer exchange. When diafiltration is used for sample desalting or buffer exchange, there is no resulting change in buffer composition. A solution volume with 100 mM salt still contains 100 mM salt after the initial concentration spin. Rediluting the retentate with water and spinning again effectively decreases the salt concentration of the sample by the concentration factor of the ultrafiltration. For example, if a 4,000 L sample containing 100 mM salt is concentrated to 50 L (80X) in an Amicon Ultra centrifugal filter unit, rediluted with water to 4,000 L, and reconcentrated, the salt concentration will be reduced 80X to 1.25 mM. To achieve more complete salt removal, multiple concentration and redilution spins are required. For most samples, two concentration/reconstitution cycles will remove about 99% of the initial salt content. With very small sample volumes, dilution of the sample before the initial concentration spin can often decrease salt concentration to an acceptable level. For example, if a 200 L sample containing 100 mM salt is diluted to 4,000 L before concentration in an Amicon Ultra centrifugal filter unit, the salt concentration in the 4,000 L sample will be 5 mM. The concentrate will still contain 5 mM salt. If more complete salt removal is desired, a re-dilution/spin cycle should be added. In this example, if the original spin ended with 50 L of retentate, redilution to 4,000 L results in 0.06 mM salt concentration. The sample can then be reconcentrated to 50 L in an Amicon Ultra centrifugal filter device. Diafiltration can be a continuous or a discontinuous process. In continuous diafiltration, such as in a stirred cell or a TFF device, the solution is maintained at a fixed volume while solvent flows continuously through the mixture. Salts and other microsolutes are steadily removed by convective transport. Microsolute exchange can be accomplished using the same principle. Constant operator attention is not required and the possibility of solute denaturation by overconcentration is eliminated. In discontinuous diafiltration, such as in a centrifugal ultrafiltration device, salts and microsolutes are removed by repeated concentration and dilution (Figure 5).
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
10 L
90 L
90 L
100 mM NaCl
100 mM NaCl
10 mM NaCl
10 mM NaCl
10
Transport convective with solvent, independent of microsolute composition. Rapid rate. Fractional removal independent of content. Ultrafiltration rate reduced with decreased temperature (net effect not as marked). At elevated macrosolute content, ultrafiltration rate reduced. Minimal exchange solvent required; easily contained in reservoir. Simple automation with endpoint control apparatus.
Transport diffusion-controlled, dependent on type of microsolute. Slow transport. Lower efficiency with decreased microsolute concentration. Marked temperature dependence (reduced transport at lower temperature). Microsolute transport relatively unaffected by macrosolute content. Frequent dialysate change. Recirculation about bags to maximize transport. Automation possible with complex equipment.
Fractionation
Fractionation is the process of separating a mixture into its components using a combination of physicochemical properties of the solute. Ultrafiltration membranes have been used for fractionation of protein solutions on the basis of size1. This technique is also called membrane partitioning chromatography1,2. Here, proteins larger than the pore size are retained and those smaller than the pore size pass through into the permeate. Since fractionation is rarely absolute, acceptability of the results will depend on whether the passing solute, retained solute or both are of interest to the researcher and their levels of purity and yield. Hence fractionation efficiency is defined as a function of yield and purity relative to the starting solution3.
Example separation: Separating a 10 kDa species from a 40 kDa protein is very likely to succeed using a 30K MWCO membrane. This is due to the fact that the retained solute is >0.9X of the MWCO. Secondly the size difference between the solutes is 4X and the ratio of the MWCO to the passing solute is also 4X.
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In the case of separating an 8 kDa from a 24 kDa using a 30K MWCO, fractionation will not be successful because the 24 kDa species is only 80% of the pore size of the 30K MWCO and it may leak into the permeate. Thus, careful attention needs to be paid to the size of the solutes and the available MWCOs. If the application requires the retained species, then the rules are reversed. For maximal purity, the MWCO chosen should be very open, and for maximal yield, the MWCO should be very tight. 2 . Starting concentration of proteins: Starting concentrations of protein solutions affect fractionation efficiency4,5. When fractionating with an Amicon Ultra device, the starting concentration of the passing solute, to 10 mg/ mL does not affect yields or purity of the solute in the permeate fraction. At the other end, for dilute proteins, where the concentrations are below 0.5 mg/mL, the polarizing gel-like layer does not foul the membrane. In this case we recommend using the lowest MWCO possible to prevent any trace amounts of retained solute appearing in the filtrate. Example separation: Consider the separation of a 17 kDa protein from a 66 kDa protein. If a MWCO of 50K is chosen (ratio of MWCO to passing ~3X), yields and purity of the passing solute are not affected even when the starting concentration of the retained is greater than 5 mg/mL. However, if the 30K MWCO is chosen to maximize purity of the passing solute, then yields are significantly affected as concentration of the retained increases to 5 mg/mL and beyond. This is due to the fact that the ratio of the MWCO to the passing solute is ~1.8X. 3 . Multiple diafiltration steps to increase yields: In order to increase yields of the passing solute and to increase purity of the retained solute, multiple spins are necessary. This approach is also called as diafiltration, where the retentate fraction is diluted back to the starting volume and the sample is centrifuged again. For e.g., if 50% of the passing solute was recovered in the filtrate in the first spin, 50% of the remaining, i.e., 25% would be recovered in a subsequent spin. The total for 3 spins would be 50 + 25 + 12.5,
i.e. 87.5%. This relation holds true for the most part since the total amount of the retained solute is unchanged. With multiple spins, more of the passing solute goes through into the permeate, leaving the retained fraction purer and increasing the yield of the passing solute. 4 . A serial fractionation strategy for compartmentalizing unknown or complex mixtures: Our recommendation for fractionation of unknown mixtures is to start by separating the proteins using the highest MWCO available. The permeate fraction from this separation is fractionated on the next highest MWCO and so on, serially. Thus, the permeate fraction from a device with a 100K MWCO membrane contains proteins <100 kDa and has undetectable amounts of proteins with higher molecular weights. Similarly, the permeate from the 50K MWCO contains proteins <50 kDa and so on. A point to note is that the efficiency of removing proteins higher than the cutoff depends on the size difference between the solute and the MWCO, and the starting concentration of that solute. Smaller solutes will pass through preferentially compared to the larger ones.*
References
1. Blatt WF, Hudson BG, Robinson SM, Zipilivan EM. Fractionation of protein solutions by membrane partition chromatography. Nature 1967;(216)511-513. 2. Blatt WF. Membrane partition chromatography: a tool for fractionation of protein mixtures. J Agric Food Chem 1971;19:589-594. 3. Ngiam SH, Bracewell DG, Zhou Y, TitchenerHooker NJ. Quantifying process tradeoffs in the operation of chromatographic sequences. Biotechnol Prog 2003;19:1315-1322. 4. Robertson BC, Zydney AL. Polarization and adsorption effects on sieving in membrane protein filtration. ASAIO Trans 1987;33: 118-122. 5. Nel RG, Oppenheim SF, Rodgers VG. Effects of solution properties on solute and permeate flux in bovine serum albumin-IgG ultrafiltration. Biotechnol Prog 1994;10:539-542.
*Refer to A Simple Strategy for Protein Fractionation Using Ultrafiltration for a protocol.
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Membrane Selection . . . . . . . . . . . . . . . . . . . 14 Device Selection . . . . . . . . . . . . . . . . . . . . . . 16 Microcon Centrifugal Filters . . . . . . . . . . . . . . 17 Amicon Ultra-4 Centrifugal Filters . . . . . . . . . . 18 Amicon Ultra-15 Centrifugal Filters . . . . . . . . . 20 Centriprep Centrifugal Filters . . . . . . . . . . . . . 22 Centricon Plus-70 Centrifugal Filters . . . . . . . . . 23 MultiScreen Filter Plate . . . . . . . . . . . . . . . . . . 24 Ultrafree Centrifugal Filters . . . . . . . . . . . . . . . 25 Pellicon XL Cassettes and Labscale TFF System . . . . . . . . . . . . . . . . . . . 26 Pellicon 2 Mini Cassettes . . . . . . . . . . . . . . . . 28 Prep/Scale Spiral Wound Filter Cartridges . . . . 29 Stirred Cells . . . . . . . . . . . . . . . . . . . . . . . . . 30 Ultrafiltration Discs . . . . . . . . . . . . . . . . . . . . . 31
GUidbrT o ss laend in GvM eeM bro rne s arnd ilT r aTe o n M e m b r a n e P ro ce s s e s M e M e a ne e C T d e iC s f a UlT a f d e v iC i s
13
Membrane Selection
Millipore offers a complete range of centrifugal devices used for sample concentration, purification, and desalting or buffer exchange of soluble macromolecules. Millipore products are also available for applications such as cleaning up PCR reactions, separating protein-bound from free ligands, removing restriction enzymes, and recovering oligonucleotides from agarose gels. This section provides information to aid in choosing the correct product for a particular application. Millipore offers three distinct types of membranes to choose from. This section will describe these three types of membranes and then provide information about choosing the correct membrane based on typical recoveries or application.
Types of Membranes
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
Ultracel Ultrafiltration Membrane
To concentrate or desalt dilute solutions, use Ultracel series regenerated cellulose ultrafiltration membranes. The hydrophilic, tight microstructure of Ultracel membranes assures the highest possible retention with the lowest possible adsorption of protein, DNA or other macromolecules.
Q P
Q Q Q P
Q Q Q Q P
Rule of Two
For Ultracel (regenerated cellulosic) membranes, Millipore recommends using a membrane with a NMWL at least two times smaller than the molecular weight of the protein solute that one intends to concentrate.
Rule of Three
For Biomax (polyethersulfone) membranes for stirred cells and TFF, Millipore recommends using a membrane with a NMWL at least three times smaller than the molecular weight of the protein solute that one intends to concentrate.
14
Specialty Devices
Protein concentration Protein purification/desalting/buffer exchange 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
Micropure-EZ
Montage PCR
Ultracel
Biomax
100K
0.45
0.65
30K
50K
10K
0.2
5.0
0.1
3K
5K
*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).
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
15
Device Selection
See Table 3 to determine which centrifugal product to use for protein concentration based on initial sample molecular weight and volume.
Millipore Device
Small Volume Filtration Devices Microcon Centrifugal Filters* Ultrafree-MC Centrifugal Filters MultiScreen Filter Plate with Ultracel Membrane** Medium Volume Filtration Devices Ultrafree-CL Centrifugal Filters Amicon Ultra-4 Centrifugal Filters Amicon Ultra-15 Centrifugal Filters Centriprep Centrifugal Filters Large Volume Filtration Devices Centricon Plus-70 Centrifugal Filters Amicon Stirred Cells Series 8000, High Output, Solvent-Resistant Stirred Cells Tangential Flow Filtration (TFF) Pellicon XL 50 Ultrafiltration Devices Prep/Scale Spiral Wound UF Modules
U M U M U U U U U U U U
*Bold type indicates recommended devices. **96-well plate. Volume per well.
16
Microcon centrifugal filters are the lab standard for small volume concentration. They allow you to process macromolecular solutions up to 500 L using any centrifuge that can 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%
Highest Recovery
Cytochrome c (0.25 mg/mL) -Chymotrypsinogen (1 mg/mL) Ovalbumin (1 mg/mL) Bovine Serum Albumin (1 mg/mL) Bovine IgG Fraction II (1 mg/mL)
94
95 95
95 95
95 95
95
Protocol
Add Sample
Ordering Information
Description NMWL Qty/Pk* Catalogue No.
42403 42404 42406 42407 42409 42410 42415 42416 42412 42413
50,000 100,000
Recover Sample
0.5
mL
1 4 m L
Deadstop prevents sample from spinning to dryness and eliminates the need for an inverse spin -marked for in vitro diagnostic use (10K NMWL)
Comparative Performance
100
Recovery (%)
80 60 40 20 0
Brand V
Brand PG
Brand P
Amicon Ultra-4
Spin Time (min) Complex sample volumes of 4 mL can be concentrated or diafiltered in as few as 15 minutes.
18
Retentate Recovery (%) by Nominal MW Solute (Concentration) MW 3,000 10,000 30,000 50,000 100,000
94
95
95
94
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.
Add Sample
Amicon Ultra-4 Centrifugal Filters are assembled with centrifuge tubes and caps
3,000
8 24 96 8 24 96 8 24 96 8 24 96 8 24 96
UFC8 003 08 UFC8 003 24 UFC8 003 96 UFC8 010 08 UFC8 010 24 UFC8 010 96 UFC8 030 08 UFC8 030 24 UFC8 030 96 UFC8 050 08 UFC8 050 24 UFC8 050 96 UFC8 100 08 UFC8 100 24 UFC8 100 96
10,000
30,000
50,000
100,000
1 4 m L
15
mL
Comparative Performance
100
Recover (%)
80 60 40 20 0
Brand V
Brand PG
Spin Time (min) Complex sample volumes of 15 mL can be concentrated or diafiltered in as few as 15 minutes.
20
Solute (Concentration)
MW
3,000
10,000
30,000
50,000
100,000
91
93
98
93
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.
Add Sample
Amicon Ultra-15 Centrifugal Filters are assembled with centrifuge tubes and caps
3,000
8 24 96 8 24 96 8 24 96 8 24 96 8 24 96
10,000
30,000
UFC9 030 08 UFC9 030 24 UFC9 030 96 UFC9 050 08 UFC9 050 24 UFC9 050 96 UFC9 100 08 UFC9 100 24 UFC9 100 96
50,000
100,000
15
mL
5 15 m L
Cytochrome c (0.25 mg/mL) -Chymotrypsinogen (1 mg/mL) Ovalbumin (1 mg/mL) Bovine Serum Albumin (1 mg/mL) Bovine IgG Fraction II (1 mg/mL)
90
85 90
90 90
90
Protocol
Disassemble Device and Add Sample Reassemble and Place in Centriprep Centrifuge Until Equilibrium is Reached
Ordering Information
Description Membrane NMWL Qty/Pk* Catalogue No.
24 96 24 96 24 96 24 96
22
The Centricon Plus-70 device can concentrate most 70 mL solutions down to 350 L in less than 25 minutes, making it a convenient alternative to stirred cells. Typical sample recoveries are >90% with minimal sample loss due to non-specific binding. High concentration factors, with samples typically concentrated in the 50X to 200X range Low hold-up volume Invert spin method of recovery Low binding Ultracel regenerated cellulose membrane
Cytochrome c (0.25 mg/mL) Bovine Serum Albumin (1 mg/mL) IgG Fraction II (1 mg/mL)
85
94 95 94
96 91
84 91
Protocol
Add Sample
60 40 20 0 0 PBS 0.25 mg/mL BSA 1.0 mg/mL BSA 5.0 mg/mL BSA
10
20
30
40
Time (min) 5K NMWL Biomax membrane spun at 3500 x g in a swinging bucket rotor.
Ordering Information
Description Membrane NMWL Qty/Pk* Catalogue No.
Biomax Ultracel
8 8 8 8
08 08 08 08
30 70 m L
< 0.5
mL
10,000
95
10,000
99
10
20
30
40
50
60
Spin Time (min) 300 L sample spun at 2000 x g in swinging bucket rotor.
Ordering Information
Description Qty/Pk* Catalogue No.
10
MAUF 010 10
Description
Membrane
Sterility
Qty/Pk
Catalogue No.
Durapore (PVDF)
0.1 0.22
UFC3 0VV 25 UFC3 0VV 00 UFC3 0GV 25 UFC3 0GV 0S UFC3 0GV 05 UFC3 0HV 25 UFC3 0HV 00 UFC3 0HV 0S UFC3 0DV 25 UFC3 0DV 00 UFC3 0DV 0S UFC3 0SV 00 UFC3 0LG 25 UFC3 0LH 25 UFC4 0VV 25 UFC4 0VV 00 UFC4 0GV 25 UFC4 0GV 00 UFC4 0HV 25 UFC4 0HV 00 UFC4 0DV 25 UFC4 0SV 25 UFC4 0LG 25 UFC4 0LH 25
0.45
Ultrafree-CL
0.65
Protocol
Add Sample Ultrafree-CL Centrifugal Filters
Non-sterile Non-sterile Non-sterile Non-sterile Non-sterile Non-sterile Non-sterile Non-sterile Non-sterile Non-sterile
Particulates Filtrate
Collect Sample
Ordering Information
0.12 m L
12 L
Protein Concentration (g/L) Pellicon XL cassettes provide rapid, efficient concentration of protein-containing solutions. Data show concentration of BSA with a 10K NMWL cassette with Ultracel regenerated cellulose membrane on a LabScale TFF system operating at 13 C with 40 psi inlet pressure and 20 psi outlet pressure.
26
Description
Membrane
NMWL
Catalogue No.
Pellicon XL Cassettes
5,000 10,000 30,000 300,000 1,000,000 5,000 8,000 10,000 30,000 50,000 100,000 300,000 500,000 1,000,000
Pore Size (m)
PXC0 05C PXC0 10C PXC0 30C PXC3 00C PXC0 1MC PXB0 05A PXB0 08A PXB0 10A PXB0 30A PXB0 50A PXB1 00C PXB3 00C PXB5 00C PXB0 1MC
50 50 50 50 50 50 50 50 50 50 50 50 50 50
Biomax Polyethersulfone
Description
Membrane
Catalogue No.
Pellicon XL Cassettes
Durapore PVDF
50 50 50 50
Description
Catalogue No.
System kit includes pump module, stir base, 500 mL and 100 mL reservoirs, stand for 100 mL reservoir, and multi-manifold. Labscale TFF System 115 V 230 V GB-230 V XX42 LSS 11 XX42 LSS 12 XX42 LSS 13
Multi-Manifold accessory allows up to three cassettes to be mounted on the Labscale system for processing multi-liter samples.
12 L
Ordering Information
10
Pellicon 2 Mini-Cassettes
Cassette-style Pellicon 2 modules are available with Durapore PVDF (microporous) and Ultracel regenerated cellulose and Biomax polyethersulfone (ultrafiltration) membranes for high performance in large volume concentration and diafiltration applications, as well as scale-up applications. Cassettes for low viscosity (A-screen), low to medium viscosity (C-screen), and high viscosity/ high concentration (V-screen) solutions. Larger Pellicon cassette systems are available for processing 250 L or more. Contact Millipore or visit our web site for more information. Stainless steel Pellicon 2 Mini-Cassette Holder can operate up to three mini-cassettes.
Ordering Information
Description Membrane NMWL A Screen C Screen V Screen
Pellicon 2 Mini-Cassettes
Biomax Polyethersulfone
5,000 8,000 10,000 30,000 50,000 100,000 300,000 500,000 1,000,000 5,000 10,000 30,000 100,000 300,000 1,000,000
01 01 01 01 01 01
P2B0 50C 01 P2B1 00C 01 P2B3 00C 01 P2B5 00C 01 P2B0 1MC 01 P2C0 05C 01 P2C0 10C 01 P2C0 30C 01 P2C1 00C 01 P2C3 00C 01 P2C0 1MC 01
P2B0 05V 01 P2B0 08V 01 P2B0 10V 01 P2B0 30V 01 P2B0 50V 01 P2B1 00V 01 P2B3 00V 01 P2B5 00V 01 P2B0 1MV 01 P2C0 05V 01 P2C0 10V 01 P2C0 30V 01 P2C1 00V 01 P2C3 00V 01 P2C0 1MV 01
Accessories
Description Catalogue No.
28
0.1 100
0.23 150
0.54 250
Ordering Information
Description Membrane NMWL Prep/Scale-TFF-1 Prep/Scale-TFF-2 Prep/Scale-TFF-6
Regenerated Cellulose
1,000 3,000 5,000 10,000 30,000 100,000 300,000 10,000 30,000 50,000 100,000 300,000
CDUF 001 LA CDUF 001 LB CDUF 001 LC CDUF 001 LG CDUF 001 LT CDUF 001 LH CDUF 001 LM CDUF 001 TG CDUF 001 TT CDUF 001 TQ CDUF 001 TH CDUF 001 TM
CDUF 002 LA CDUF 002 LB CDUF 002 LC CDUF 002 LG CDUF 002 LT CDUF 002 LH CDUF 002 LM CDUF 002 TG CDUF 002 TT CDUF 002 TQ CDUF 002 TH CDUF 002 TM
CDUF 006 LA CDUF 006 LB CDUF 006 LC CDUF 006 LG CDUF 006 LT CDUF 006 LH CDUF 006 LM CDUF 006 TG CDUF 006 TT CDUF 006 TQ CDUF 006 TH CDUF 006 TM
Polyethersulfore
Accessories
Description Catalogue No.
XX42 PS0 01
for TFF-1 use XX8200 for TFF-2 and TFF-6 use XX80 EL
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
29
Stirred Cells
Concentrate, diafilter, and exchange buffers for macromolecule solutions including proteins, enzymes, antibodies and viruses.
M e M e a ne e C T d e iC s f a UlT a f d e v iC i s GUidbrT o ss laend in GvM eeM bro rne s arnd ilT r aTe o n M St i rreadn e ePl ro ce s s e s embr C ls
Maximum Process Volume (mL) Minimum Process Volume (mL) Membrane Diameter (mm) Effective Membrane Area (cm )
2
Ordering Information
Description Catalogue No.
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
Ultracel Ultrafiltration Discs
Regenerated Cellulose
Filter Diameter (mm) NMWL Qty/Pk 1,000 3,000 5,000 10,000 30,000 100,000
10 10 10 10 10 5 5
PLAC 025 10 PLAC 043 10 PLAC 047 10 PLAC 062 10 PLAC 076 10 PLAC 090 05 PLAC 150 05
PLBC 025 10 PLBC 043 10 PLBC 047 10 PLBC 062 10 PLBC 076 10 PLBC 090 05 PLBC 150 05
PLCC 025 10 PLCC 043 10 PLCC 047 10 PLCC 062 10 PLCC 062 10 PLCC 090 05 PLCC 150 05
PLGC 025 10 PLGC 043 10 PLGC 047 10 PLGC 062 10 PLGC 076 10 PLGC 090 05 PLGC 150 05
PLTK 025 10 PLTK 043 10 PLTK 047 10 PLTK 062 10 PLTK 076 10 PLHK 090 05 PLTK 150 05
PLHK 025 10 PLHK 043 10 PLHK 047 10 PLHK 062 10 PLHK 076 10 PLHK 090 05 PLHK 150 05
10 10 10 10 10 5 5
PBCC 025 10 PBCC 043 10 PBCC 047 10 PBCC 062 10 PBCC 076 10 PBCC 090 05 PBCC 150 05
PBGC 025 10 PBGC 043 10 PBGC 047 10 PBGC 062 10 PBGC 076 10 PBGC 090 05 PBGC 150 05
PBTK 025 10 PBTK 043 10 PBTK 047 10 PBTK 062 10 PBTK 076 10 PBTK 090 05 PBTK 150 05
PBQK 025 10 PBQK 043 10 PBQK 047 10 PBQK 062 10 PBQK 076 10 PBQK 090 05 PBQK 150 05
PBHK 025 10 PBHK 043 10 PBHK 047 10 PBHK 062 10 PBHK 076 10 PBHK 090 05 PBHK 150 05
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
31
32
GUid e T o s e l e C T in G M e M br a ne s a nd d e v iC e s
Concentration, Desalting and Buffer Exchange . 34 Detergent Removal . . . . . . . . . . . . . . . . . . . . 38 Two-Dimensional Electrophoresis Sample Preparation . . . . . . . . . . . . . . . . . . . . 40 Purification of Serum Peptides for Biomarker . . 42 Rapid Antibody Concentration . . . . . . . . . . . . 45 Affinity Purification . . . . . . . . . . . . . . . . . . . . 46 Removal of Unincorporated Label from Labeled Protein . . . . . . . . . . . . . . . . . . . 50 Rapid Purification of Monoclonal Antibodies . . 52 A Simple Strategy for Protein Fractionation . . . 54 Urine Concentration . . . . . . . . . . . . . . . . . . . . 56 Use of Centrifugal Filter Devices as an Alternative to Stirred Cells . . . . . . . . . . . . . . . 58
P ro T o C o l s f o r P ro T e in s
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Concentration, Desalting, and Buffer Exchange with Amicon Ultra or Microcon Centrifugal Filters
Introduction
Amicon centrifugal devices from Millipore are ideal for removal or exchange of salts, sugars, nucleotides, and non-aqueous solvents, as well as other materials of low molecular weight. They also serve to separate free from bound species. Millipore centrifugal concentrators provide fast, convenient, high-recovery alternatives to dialysis and ethanol precipitation. Sample dilution, often associated with spin columns, is not a problem. Salt transfer across the membrane is efficient and independent of microsolute concentration or size. Millipores Amicon Ultra-4 and -15 centrifugal filters are designed for high speed with high recovery. The devices incorporate low-binding Ultracel regenerated cellulose ultrafiltration membrane for sample concentration and purification of solutions containing dilute or purified protein solutes, antigens, antibodies, enzymes, or microorganisms. Their speed and excellent recovery make them ideal for desalting and buffer exchange applications. One of the most common applications for Amicon Ultra devices is concentration and desalting of column fractions during protein purification by various chromatography methods. Two examples below demonstrate the use of Amicon Ultra devices for high protein and enzymatic activity recovery.
Method
1. Select the device with the appropriate NMWL and volume for the application. 2. Add the sample to the reservoir of the centrifugal device. 3. If the sample is smaller than the maximum volume, it can be diluted up to the maximum volume before the first centrifugation step. This will help increase the salt removal. 4. Centrifuge at the specified g-force for the recommended amount of time. 5. Remove the initial filtrate from the filtrate tube and set aside. 6. Add enough buffer or water to the device to bring the sample volume up to 4 or 15 mL. 7. Centrifuge again. 8. Set aside the filtrate. 9. Recover the concentrated, de-salted sample. NOTE: Both of the filtrates should be retained until the concentrated sample has been analyzed.
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
Results
The transfer of salts across a membrane filter is independent of sample concentration or size. There is no change in the composition of the buffer when desalting using ultrafiltration. For example, a solution containing 500 mM salt still contains that concentration after the initial centrifugation. Adding another volume of salt-free buffer or water to the retentate and centrifuging again will reduce the salt concentration. This process, known as diafiltration, can be repeated to achieve maximum salt removal. Diafiltration can also be used when it is desirable to have the sample in a different buffer. The sample is concentrated and then repeatedly diluted with the desired buffer and concentrated again.
34
As the results show in Tables 1 and 2, the efficient design of the Millipore devices allowed >90% of the salt to be removed during the first centrifugation step. Typically, only one subsequent centrifugation step was needed to increase the typical salt removal to 99% with >90% recovery of the sample. Protein purification by chromatography usually involves the collection of multiple column fractions, with only some of those fractions containing the protein of interest. After the fractions are combined, a protein concentration step is often required for protein storage, or concentration with buffer ex-change may 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 0.25 mg/mL NMWL 5 kDa Cytochrome c 0.25 mg/mL 10 kDa BSA 1 mg /mL 30 kDa BSA 1 mg/mL 50 kDa IgG 1 mg/mL 100 kDa
1 2
94.5 92.6
97.2 99.9
97.3 95.6
97.9 99.9
96.0 94.4
98.2 99.9
98.9 92.4
97.1 99.9
99.9 97.1
97.7 99.5
Three Amicon Ultra-15 devices of each cut-off were tested with 15 mL of solute. 500 mM NaCl was added to each solution. Each spin was performed at 4000 x g for 30 minutes. After the first spin, the retentate was brought up to 15 mL with ultrapure water from a Milli-Q (Millipore) system. OD readings were taken at 410 nm for Cytochrome c and 280 nm for BSA and IgG.
Table 2 . Removal of riboflavin and recovery of IgG with Microcon filter device with Ultracel membrane
Spin Number % NaCI Remaining % IgG Recovered
1 2 3 4
9 2 <1 <1
95 93 93 92
500 L of a 50:50 mixture of riboflavin and IgG were spun in a Microcon 3K NMWL device at 12,000 x g for 75 minutes at room temperature in 55 angle rotor. After the initial spin, the retentate was twice diluted with 500 L of PBS and spun again. After each spin, concentration of riboflavin and IgG in the filtrate and retentate were monitored.
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
35
Spin
% Protein Recovery
% NaCl Removal
% Protein Recovery
% NaCl Removal
% Protein Recovery
% NaCl Removal
% Protein Recovery
% NaCl Removal
% Protein Recovery
% NaCl Removal
Recombinant IDO was expressed in E. coli BL21 (DE3) cells utilizing the pET 28a (+) vector system. In this system, a hexahistidyl tag was fused to fullsize IDO at the N-terminus with a spacer sequence and a thrombin cleavage site. The protein was purified by conventional His-tag purification methods and eluted with imidazole. The histidine tag was removed by thrombin cleavage. Final purification was done by gel filtration chromatography G-75. Amicon Ultra-15 centrifugal devices were used to concentrate the IDO fractions from an initial concentration of ~0.5 mg/mL to a final concentration of 10 mg/mL. Samples were analyzed by SDS-PAGE using a 12.5% polyacrylamide gel (Figure 1). In addition, it was shown that no IDO activity loss was observed after concentration using an Amicon Ultra device.
developed for PKR, and PKR has been purified using three chromatography steps on Agarose-Heparin, Agarose-Poly (I), Poly (C) and Sephacryl S-200 gel filtration columns. After the last step, PKR-containing column fractions were pooled and concentrated using Amicon Ultra-15 30K NMWL devices. The concentration step was necessary for long-term protein storage. Table 3 shows the protein recovery results obtained after four concentrations. Over 90% recovery was obtained and no protein loss to the filtrate was observed. Amicon Ultra-15 30K NMWL devices were also used for exchanging buffer for PKR autophosphorylation (activation) assay. 200 L of 6.301 mg/mL PKR in Protein Storage buffer (20 mM HEPES, 1 M NaCl, 10 mM b-mercaptoethanol, 0.1 mM EDTA, 10% glycerol, pH 7.5) was diluted to 15 mL with Phosphorylation Buffer (20 mM HEPES, 50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, pH 7.5) and re-concentrated three times using
Figure 1 .
40 kDa 35 kDa
SDS-PAGE of purified indoleamine 2,3-dioxygenase before and after concentration using Amicon Ultra-15 centrifugal devices.
Starting Volume (mL) Starting Concentration (mg/mL) Volume of Retentate (L) Total Amount of PKR (mg) Before UF After UF Filtrate % Recovery
36
Amicon Ultra devices at 3000 x g for 20 minutes at 4 C. The filtrates from the three steps were pooled and the total amount of protein in all samples was determined by UV absorption A280. Protein activity was tested by autophosphorylation assay. The protein in the storage buffer was supplemented with 5 mM MgCl2 and the samples were allowed to undergo autophosphorylation at 30 C for 20 minutes in the presence of 3 mM ATP and 3 Ci [g32P] ATP. The activity was determined by autoradiography and quantified by liquid scintillation counting. As shown in Figure 2, the activity of PKR when no buffer exchange was only 6% of that when buffer exchange step was performed. Hence, the UF successfully exchanged the buffer while maintaining the activity of the protein.
Figure 2 .
100
80 60 40 20 0
No Buffer Exchange
Buffer Exchange
Comparison of PKR autophosphorylation with buffer exchange by ultrafiltration and without buffer exchange.
References
1. Samuel CE. Antiviral actions of interferon. Interferon-regulated cellular proteins and their surprisingly selective antiviral activities. Virology 1991;183(1):111. 2. Hovanessian AG. The double stranded RNAactivated protein kinase induced by interferon: dsRNA-PK. J Interferon Res 1989;9(6):641 7. 3. Lebleu B, et al. Interferon, double-stranded RNA, and protein phosphorylation. Proc Natl Acad Sci USA 1976;73(9):310711. 4. Samuel CE. Mechanism of interferon action: phosphorylation of protein synthesis initiation factor eIF-2 in interferon-treated human cells by a ribosome-associated kinase processing site specificity similar to hemin-regulated rabbit reticulocyte kinase. Proc Natl Acad Sci USA 1979;76(2):6004. 5. Koromilas AE, et al. Malignant transformation by a mutant of the IFN-inducible dsRNAdependent protein kinase. Science 1992; 257:16859. 6. Meurs E, et al. Tumor supressor function of interferon-induced double-stranded RNA activated protein kinase. Proc Natl Acad Sci USA 1993;90:2326. 7. Wong AH, et al. Physical association between STAT1 and the interferon-inducible protein kinase PKR and implications for interferon and double-stranded RNA signaling pathways. EMBO J, 1997;16(6):1291304. 8. Cuddihy AR, et al. Double-stranded-RNAactivated protein kinase PKR enhances transcriptional activation by tumor suppressor p53. Mol Cell Biol 1999;19(4):247584. 9. Demarchi F, Gutierrez MI, Giacca M. Human immunodeficiency virus type 1 Tat protein activates transcription factor NF-kappaB through the cellular interferon-inducible, doublestranded RNA-dependent protein kinase, PKR. J Virol 1999; 73(8):70806. 10. Petryshyn R, et al. Effect of interferon on protein translation during growth stages of 3T3 cells. Arch Biochem Biophys 1996;326(2):2907. 11. Barber GN. Host defense, viruses and apoptosis. Cell Death Differ 2001;8(2):11326. 12. Tan SL, Katze MG. The emerging role of the interferon-induced PKR protein kinase as a apoptotic effector: A new face of death? J Interferon Cytokine Res 1999;19:54354. 13. Balachandran S, et al. Activation of the dsRNA-dependent protein kinase, PKR, induces apoptosis through FADD-mediated death signaling. EMBO J, 1998;17(23):6888902. 14. Osman F, et al. A cis-acting element in the 3untranslated region of human TNF-alpha mRNA renders splicing dependent on the activation of protein kinase PKR. Genes Dev 1999;13(24): 328093.
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
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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 Microcon centrifugal devices
NMWL Detergent (%) 3 kDa 10 kDa 30 kDa 50 kDa 100 kDa
Table 2 . Percent detergent removal after one spin with Amicon Ultra-4 centrifugal devices
NMWL Detergent (%) 10 kDa 30 kDa
>90% >90% 4089% <40% >90% >90% 4089% >90% >90% 4089% <40% <40% >90% <40% <40% >90% 4089% <40% <40% <40% <40% <40% 4089% <40% <40% <40% >90% 4089% <40%
>90% >90% 4089% <40% >90% >90% 4089% >90% >90% 4089% <40% <40% >90% <40% <40% >90% 4089% <40% <40% <40% <40% <40% 4089% <40% <40% <40% >90% 4089% <40%
>90% >90% 4089% <40% >90% >90% 4089% >90% >90% 4089% <40% <40% >90% <40% <40% >90% 4089% <40% 4089% <40% <40% <40% 4089% <40% <40% <40% >90% >90% 4089%
>90% >90% 4089% <40% >90% >90% >90% >90% >90% 4089% <40% <40% >90% 4089% <40% >90% 4089% <40% 4089% 4089% <40% <40% 4089% <40% <40% <40% >90% >90% >90%
>90% >90% 4089% <40% >90% >90% >90% >90% >90% 4089% <40% <40% >90% <90% <90% >90% 4089% <40% >90% >90% 4089% <40% >90% >90% 4089% <40% >90% >90% >90%
74% 13% 0.5% 47% 36% 39% 40% 26% 37% 96% 62% 36%
75% 11% 2.0% 69% 42% 47% 70% 37% 39% 81% 96% 80%
TDMABr2 0.1 1 5 Digitonin 0.01 0.1 1 Tween-20 0.01 0.1 1 5 Triton X-100 0.01 0.1 1 5 CHAPS 0.1 1 5
1 2
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
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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
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.
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Figure 2 .
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.
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
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Purification of Serum Peptides for Biomarker Research with Amicon Ultra Centrifugal Filters
Introduction
A biomarker can be defined as a molecule that indicates an alteration in physiology. Biomarkers play an essential role in the drug discovery and development process. They provide powerful clues to genetic susceptibility, disease progression, and predisposition, as well as drug response and the physiological and metabolic profiles of diseases and drug responses. Biomarkers can also provide valuable diagnostic and prognostic information that can facilitate personalized medicine. Peptide and protein patterns have been linked to ovarian cancer, breast cancer, prostate cancer and astrocytoma15. Most diagnostic tests are based on blood or urine analysis5. Serum is a key source of putative protein biomarkers, and, by its nature, can elucidate organ-confined events. Mass spectrometry, coupled with bioinformatics, is capable of distinguishing between serum protein pattern signatures in latestage and early-stage ovarian cancer patients6. One of the major impediments to the discovery of new biomarkers is the presence of salts, proteins, and lipids in plasma or serum that makes it difficult to detect and analyze peptides by mass spectrometry. When untreated serum is spotted onto a MALDI-TOF plate, it does not produce any usable signal in mass spectrometry. Multiple protocols have been developed to extract and enrich peptides from tissues and body fluids, such as batch reversed phase chromatography over C18 resin and extraction with 0.1% trifluoroacetic acid (TFA) or 50% acetonitrile to selectively precipitate large proteins while enhancing the solubility of smaller proteins and peptides. Ultrafiltration has previously been reported as a sample preparation tool to prepare low molecular weight fractions for biomarker analysis710. In this study we show that ultrafiltration in combination with solid phase extraction (SPE) on C18 resin can be a convenient and efficient method for serum peptide purification. The approach provides more peptides for mass spectrometry analysis than the acetonitrile precipitation method.
Methods
Preparation of Serum Peptides by UF and SPE
One milliliter of human serum, with or without acetonitrile, was filtered using Amicon Ultra-4 10K NMWL centrifugal devices. The devices were centrifuged in a swinging bucket rotor for 15 to 30 minutes at 3000 x g. Ten microliters of the filtrate was acidified with 5 L of 1% TFA, concentrated with ZipTipC18 pipette tips. Co-elution was performed directly onto a MALDI target with 2 L of -cyano-4-hydroxycinnamic acid matrix (5 mg/mL in 50% acetonitrile, 0.1% TFA). If acetonitrile was added to the serum prior to the filtration, the samples were briefly evaporated in a Speed Vac centrifuge to remove solvent before ZipTip purification.
42
Results
While human serum contains numerous peptides and small proteins, they are not accessible by direct mass spectrometry analysis. Even after reverse phase concentration and desalting, only a few peptides are detectable in the mass spectrum (data not shown). This can be explained by the high concentration of proteins and lipids competing with the peptides to bind to the resin (Figure 1A).
One of the common methods for serum peptide preparation for mass spectrometry is acetonitrile fractionation, where the addition of 5070% acetonitrile precipitates larger proteins, while the peptides stay soluble in the supernatant. Another way to produce relatively protein-free filtrates is ultrafiltration. Figure 2 presents the MALDI-TOF spectra of (A) straight rat serum; (B) serum supernatant after 50% acetonitrile precipitation; and (C)
Figure 1 .
A B C
MALDI-TOF spectra of (A) unprocessed rat serum; (B) serum peptides in 50% acetonitrile supernatant; and (C) serum ultrafiltrate processed with Amicon Ultra-4 30K NMWL centrifugal device.
Figure 2 .
A B
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.
serum ultrafiltrate processed with an Amicon Ultra-4 30K NMWL device. We observed higher quality spectra and an increased number of MALDI-TOF detected peptides if the serum was ultrafiltered. Further improvement of spectra can be achieved by reverse phase chromatography, which concentrates and desalts the peptides. The ZipTipC18 pipette tip is a convenient and efficient tool for micro-scale sample preparation prior to mass spectrometry. Figure 2 shows the MALDI-TOF spectra of rat serum peptides prepared with ZipTipC18 pipette tip out of (A) straight serum, (B) 50% acetonitrile supernatant and (C) serum processed by ultrafiltration. The last method provided stronger MALDI-TOF signal, higher signal-to-noise ratio and double the number of detected peptides. The addition of 5 to 10 % acetonitrile to serum and plasma samples (data not shown) prior to ultrafiltration was shown to improve the detection of serum peptides, the MALDI spectrum and the overall signal intensity.
References
1. Ardekani AM, Liotta LA, Petricoin III. Expert Rev Mol Diagn 2002;2:12. 2. Carter D, Douglass JF, Cornellison CD, Retter MW, Johnson JC, Bennington AA, Fleming TP, Reed SG, Houghton RL, Diamond TS, Vedvick TS. Biochemistry 2002;41:6714. 3. Wellmann A, Wollscheid V, Lu H, Ma ZL, Albers P, Schutze K, Rohde V, Behrens P, Dreschers S, Ko Y, Wernert N. Int J Mol Med 2002;9:341. 4. Petricoin EF, Ardekani AM, Hitt BA, Levine PJ, Fusaro VA, Steiberg SM, Mills GB, Simone C, Fishman DA, Kohn EC, Liotta LA. Lancet 2002; 359:572. 5. Bischoff R, Luider TM. J Chrom B 2004;803: 2740. 6. Stevens EV, Liotta LA, Kohn EC. J Gynecol Cancer 2003;13:1339. 7. Schulz-Knappe P, Schrader M, Standker L, Richter R, Hess R, Jurgens M, Forssmann W-G. J Chromatogr A 1997;776:125132. 8. Basso D, Valerio A, Seraglia R, Mazzza S, Piva MG, Greco E, Fogar P, Gall N, Pedrazzoli S, Tiengo A, and Plebani M. Pancreas 2002;24:814. 9. Prazeres S, Santos MA, Ferreira HG, Sobrinho LG. Clin Endocrinol (Oxf) 2003;58:68690. 10. Tirumalai RS, Chan KC, Prieto DA, Issaq HJ, Conrads TP, Veenstra TD, Mol Cell Proteomics 2003;10:1096103. 11. Chernokalskaya E, Gutierrez S, Pitt AM, Leonard J. Electrophoresis 2004;25: 24612468. 12. Millipore Case Study PR0001EN00. Automating Multiplexed Cytokine Assays manuscript in preparation.
Conclusion
For the analysis of serum peptides, complexity reduction by eliminating higher molecular weight proteins is critical for high resolution mass spectrometry. Efficient separation of peptides from the majority of proteins and salts can be achieved by sample ultrafiltration. We have shown the effective use of Amicon Ultra-4 30K NMWL centrifugal devices for the preparation of peptides. Other molecular weight cut-offs can be utilized depending on the desired range of peptides. The method can be used directly in combination with ZipTipC18 pipette tips for peptide identification by MS/MS or as a first step prior to further surface-mediated enrichment using SELDI-TOF methods. These sample preparation protocols may also be applicable to other low molecular weight markers such as drugs and metabolites. Peptide purification using ultrafiltration 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.
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Figure 1 .
40 PROSEP-A 12 10 8 20 6 4 10 2 0 0
30
PROSEP-G
10
15
20
Centrifugation Time (min) Concentration of rabbit IgG with Amicon Ultra-15 devices. The IgGs were purified using Montage PROSEP-A or PROSEP-G Antibody Purification Kits. The lines show IgG volume reduction, while the bars show a proportional increase in IgG concentration after 20 minutes of centrifugation time.
SDS-PAGE gel of purified rabbit IgG before and after concentration with Amicon Ultra-15 devices. Lane 1: Lanes 2, 3: MW standards PROSEP-A-purified IgG before concentration (lane 2) and after concentration (lane 3) PROSEP-G-purified IgG before concentration (lane 4) and after concentration (lane 5)
kDa 116
66 55
Heavy Chain
36 Light Chain
Lanes 4, 5:
21 14
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Figure 2 .
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3. Centrifuging the resin to the bottom 4. Pipetting off the supernatant 5. Washing a few times (using steps 2 and 3) 6. Eluting with a small amount of eluant Although the method is relatively simple, care must be taken not to remove the chromatography resin when pipetting off the supernatant. Pre-packed mini-spin columns are a convenient tool for small-scale protein purification. Operated by centrifugation, they usually complete the whole procedure in less than an hour.
Another alternative for small-scale purification are centrifugal devices with microporous membrane, such as Ultrafree-MC centrifugal devices. The sample can be added to the filter basket and mixed for the needed residence time and then centrifuged. The process removes the interstitial liquid but does not dehydrate the beads. Washing and elution can also be performed in a similar manner and are more effective due to the efficient removal of buffer and/or eluant. Ultrafree-MC centrifugal filter units with microporous membrane come with low protein-binding Durapore PVDF membrane in five different pore sizes from 0.1 to 5.0 m. Affinity resin can be loaded into the filter basket and the device used as a home-made mini-spin column. We show the applicability of the device for purification of rabbit IgG on PROSEP-A resin and His-tagged C-RP protein on three different commercial metal-chelate resins.
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Materials
Ultrafree-MC 0.45 m centrifugal devices (Millipore cat. no. UFC3 0HV 00) PROSEP-A high capacity resin (Millipore cat. no. 1131 118 26) Rabbit serum Gibco (Invitrogen lot no. 1132782) Micro-centrifuge Biofuge Pico (Heraeus instruments) Jouan CR1822 fixed angle rotor centrifuge Xcell SureLock Mini-cell vertical electrophoresis system (Invitrogen cat. no. EI0001) NuPage NOVEX Bis-Tris 4 12%, 1 mm thick, 15 well SDS gels, (Invitrogen cat. no. NP0323) NuPage Sample Reducing agent (10X) (Invitrogen cat. no. NP009) NuPage SDS Sample Buffer (4X) (Invitrogen cat. no. NP007) SimplyBlue SafeStain Coomassie G-250 stain (Invitrogen cat. no. LC6060)
Procedure
1. 200 mg of PROSEP-A media were placed in Ultrafree-MC 0.45 m filter basket. 2. The columns were equilibrated with 400 L of binding buffer A and centrifuged for 1 minute at 100 x g. 3. 200 L of rabbit serum were diluted 1:1 with binding buffer and the entire volume was loaded into the spin column containing PROSEP-A resin. 4. Devices were placed on a shaker for 15 minutes at room temperature and centrifuged at 100 x g for 5 minutes. Flow-through was collected for future analysis. 5. Three consecutive washes of 400 L each were performed by adding 400 L of binding buffer A and centrifuging at 2,000 x g for 2 minutes each.
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.
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6. 200 L of elution buffer B2 were added and centrifuged for 2 minutes at 2,000 x g.
5. 500 L of the clarified lysate were added to the resin. 6. The His-tagged proteins were bound for 30 minutes with light agitation. 7. The lysate was removed by centrifugation at 500 x g for 10 minutes. 8. The resin was washed with 500 L of wash buffer for 5 minutes with agitation. The wash solution was removed by centrifugation for 5 minutes at 500 x g. This step was repeated two more times. 9. 250 L of elution buffer were added to the Ultrafree-MC device and mixed for 5 minutes. Purified protein was recovered by centrifugation at 500 x g for 1 minute.
Procedure
1. Recombinant proteins were expressed in Escherichia coli. 2. Cells were prepared at a 10X concentration using lysis buffer. Lysozyme was added to a concentration of 0.1 mg/mL. To reduce the viscosity, benzonase was added to the lysate. The lysates were clarified by centrifugation. 3. 200 L of the 50% resin slurry were added to the Ultrafree-MC device and the residual fluid was removed by centrifugation for 1 minute at 500 x g. 4. The resin was equilibrated with 500 L of binding buffer and centrifuged for 2 minutes at 500 x g.
Results
The results of purifying rabbit IgG using UltrafreeMC centrifugal devices are shown in Figure 1. The device was challenged with approximately 14 mg of total protein, with an estimated IgG content of 1.52 mg. The original serum, flowthrough, three washes and two eluted fractions were analyzed by SDS-PAGE. The total amount of purified IgG, as estimated by OD280, was 1.2 mg and 1.1 mg on two devices processed in parallel. The whole procedure was completed in less than one hour. This method can be useful for monitoring the titer of antigen-specific antibodies after immune activation, or whenever small amounts of IgG need to be purified.
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Figure 2 shows the results of His-tagged protein purification in Ultrafree-MC devices using Ni-NTA agarose and BD Talon resin. Two recombinant proteins were purified: C-RP, high-expressing protein of 26 kDa; and RT66, low-expressing protein of 66 kDa. Both purifications resulted in high purity proteins. The amount of proteins purified out of 500 L of lysate was 3235 g for C-RP protein and 8 g for RT66. The data show that affinity batch purification can be effectively performed on a small scale using Ultrafree-MC devices loaded with resin. The process combines the high efficiency of batch binding and washing with the handling convenience of a mini-spin column. With minimal handson time, the method provides flexibility of resin to lysate ratio and binding conditions, independent of centrifugation speed and rotor angle. This method is applicable to recombinant protein purification, antibody purification and immunoprecipitation.
Figure 1 .
Rabbit IgG purification on PROSEP-A resin in Ultrafree-MC devices. Lane 1: Molecular weight standards Rabbit serum Flow through
kDa 200 116.3 97.4 66.3 55.4 36.5 31.0
Lane 2: Lane 3:
Lanes 46: Three consec- 21.5 utive washes Lanes 7, 8: Eluted IgG from two devices
14.4 6.0
Figure 2 .
His-tagged protein purification using Ultrafree-MC devices. Lane 1: Molecular weight standards E. coli lysate expressing C-RP protein E. coli lysate expressing RT66 protein
kDa 200 116.3 97.4 66.3 55.4 36.5 31.0 21.5 14.4 6.0
RT66
Lane 2:
C-RP
Lane 3:
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Removal of Unincorporated Label from Labeled Protein with Amicon Ultra Centrifugal Filters
Introduction
Centrifugal devices containing ultrafiltration membranes are ideal for the removal or exchange of salts, sugars, nucleotides, non-aqueous solvents, and other materials of low molecular weight. They also serve to separate free from bound species. For the removal of unincorporated label in protein labeling applications, Millipore centrifugal devices provide fast, convenient, high-recovery alternatives to gel filtration. Sample dilution, often associated with gel filtration, is not a problem. A few rounds of diafiltration can efficiently remove unincorporated label. Two examples below demonstrate the use of Millipore centrifugal ultrafiltration devices for removal of unreacted fluorescent and isotope labels.
Results
Figure 1 shows the SDS-PAGE gel of FITC-labeled BSA before and after each of four diafiltration cycles. Unincorporated FITC is clearly visible in the starting material and in the first filtrate. After only one cycle of ultrafiltration, the majority of the free label is removed. Subsequent cycles of filtration result in BSA that is virtually free of unincorporated FITC. Fluorescence measurement offers a more sensitive method to monitor the ultrafiltration process. Figure 2 shows the change in fluorescence of filtrate and retentate during four cycles of FITC-BSA diafiltration. The results indicate that almost 80% of free FITC can be removed after the first ultrafiltration and three rounds result in 98% removal. This demonstrates the viability of ultrafiltration as an alternative to gel filtration for cleaning up protein labeling reactions. Using an Amicon Ultra-4 device, each ultrafiltration is accomplished in 1015 minutes and allows high recovery of target protein. While gel filtration results in diluted protein fractions, ultrafiltration offers the additional advantage of concentrating protein while removing unincorporated label.
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Figure 1 .
SDS-PAGE of FITC-labeled BSA Lane 1: Starting material Lane 2: Retentate after first ultrafiltration Lane 3: Retentate after 2 rounds of ultrafiltration Lane 4: Retentate after 3 rounds of ultrafiltration Lane 5: Retentate after 4 rounds of ultrafiltration Lane 6: Filtrate after first ultrafiltration Lane 7: Filtrate after 2 rounds of ultrafiltration Lane 8: Filtrate after 3 rounds of ultrafiltration
Figure 2 .
60,000 50,000 Retentate Filtrate
Starting material
Spin 1
Spin 2
Spin 3
Spin 4
FITC-BSA
FITC
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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.
Method
The method involves clarification of the hybridoma supernatant by microfiltration using a Stericup vacuum filter cup, followed by concentration using ultrafiltration1 with a Centricon Plus-70 device (see Table 1 and Figure 1 for method details). The MAb is further purified on protein A/G beads. The purified MAb is desalted and buffer-exchanged using ultrafiltration.
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
Ultrafiltration
Centrifugal Tangential Flow Filtration (TFF)
1. Start with 200 mL 2. Weigh out ammonium sulfate 3. Add ammonium sulfate to clarified supernatant with constant stirring (20-30 min). Store at 4 C. 4. Centrifuge to recover pellet, resuspend pellet 5. Prepare dialysis membrane and check for integrity 6. Collect resuspended precipitate in dialysis tubing/device 7. Dialyze with three changes of PBS (18-24 hrs) 8. Collect dialysate in centrifuge tube(s) and centrifuge (~30 min) 9. Load on protein-G column Total time required ~24 hrs
1. Start with 200 mL 2. Add supernatant to Centricon Plus-70 Device (100K MWCO) 3. Centrifuge (2030 min) 4. Invert spin to collect sample (2 min) 5. Load on protein-G column
1. Start with 1000 mL 2. Add supernatant to Labscale TFF unit (30K MWCO) 3. Apply pressure (40 psi) with constant stirring (~2 hrs) 4. Collect sample (5 min)
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Results
This approach was successfully used to purify an anti c-myc antibody secreted by the hybridoma clone 9E102 (Figure 1). The MAb purified by this new method performed comparably to the commercially 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 application of ultrafiltration to MAb purification will be of considerable value to any researcher interested in screening hybridoma libraries and accelerating the purification of MAbs.
Figure 2 .
2 Abalone Commercial
Traditional
UF-based
Figure 1 .
C: Clarified supernatant Tr: Supernatant precipitated with ammonium sulfate and dialyzed using 10K MWCO membrane (Spectrapor, Rancho Dominguez, CA) UF: Supernatant concentrated by ultrafiltration on Centricon Plus 70 device
Tr
UF
OD @ 450 mm
10
100
Concentration of mAB (g/mL) Upper panel shows activity comparison measured in a western blot between MAb (derived from HEK 293) purified using ultrafiltration method versus traditional method. Three-fold serial dilutions of HEK 293 cell nuclear extracts were run on 4 to 12% NuPAGE gels and transferred to Immobilon-P membrane (Millipore). Blots were probed with the indicated primary antibodies and secondary anti-mouse alkaline phosphatase conjugate (Sigma, St. Louis, MO). The antibodies were detected with Immobilon Western AP substrate (Millipore) and imaged on a Kodak scanner. Bottom panel shows activity comparison measured in an ELISA assay. HEK 293 cells were grown on 96-well plates. After fixation and epitope-retrieval by heating for 10 minutes in a microwave, cells were permeabilized with 1% saponin (Sigma) in PBS + 2% normal donkey serum (Jackson Immunoresearch, West Grove, PA) and treated with serial dilutions of the indicated purified MAbs. The cells were then washed and treated with goat anti-mouse HRP-conjugate antibody (Sigma). The reactions were developed using a SureBlue TMB HRP substrate (KPL, Gaithersburg, MD). The readings were measured on a SpectraMax plate reader (Molecular Devices, Sunnyvale, CA).
Conclusion
Our data show that the combination of microfiltration and ultrafiltration is a rapid method for MAb purification and that MAb purified on Montage centrifugal columns with Protein G media is comparable to the commercially available MAb in purity, activity and cost. The Ultrafiltration-based MAb purification method, compared to the traditional method, is faster (23 hours versus 2 days), easier to use, and yields higher recoveries.
References
1. Saha K, Case R, Wong PK. J Immunol Methods 1992;151(1-2):307-308. 2. Evan GI, Lewis GK, Ramsay G, Bishop JM. Mol Cell Biol 1985;5(12):3610-3616.
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Method
This experiment demonstrates the use of ultrafiltration devices to enrich both LMW and high molecular weight (HMW) fractions from serum. Protein separation was accomplished by serial filtration through decreasing molecular weight cutoff (MWCO) devices from 100K to 10K MWCO. This serial enrichment strategy enabled the com partmentalization of proteins by size as well as an increase in throughput as compared to directly enriching samples using a lower MWCO device.
P ro t o c o l s f o r P ro t e in s P ro te i n E n rich m e n t U s i n g U l t r af i l t r a t io n
Figure 2. Purification of Cytochrome c from a mixture containing 10% fetal bovine serum (FBS) and the compartmentalization of protein fractions using Amicon Ultra devices
M S R100 P100 R50 P50 R30 P30
50 kDa Retentate
30 kDa
Purified Cytochrome c
30 kDa Retentate
M: Marker S: 10% FBS + 1 mg/mL Cytochrome c R100: 100 kDa Retentate P100: 100 kDa Permeate
R50: 50 kDa Rententate P50: 50 kDa Permeate R30: 30 kDa Retentate P30: 30 kDa Permeate
A complex mixture is centrifuged in a 100K device. The retentate is removed and saved for later analysis. The permeate fraction is then centrifuged in a 50K device. This process is repeated for the 30K device.
A serum-protein mixture spiked with purified Cytochrome c (12.5 kDa) was centrifuged in an Amicon Ultra 4 mL device using serially decreasing MWCOs as described in Figure 1. The red, blue and green lines indicate approximate molecular weight ranges and represent the theoretical MWCOs. Colored dashed boxes represent size specific protein compartments.
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The traditional methodology employing use of a single MWCO to remove HMW and enrich for the LMW resulted in impure fractions compared to those from serial enrichment.
References
1. Forssmann WG, et al. J Chromatogr A 1997;776:125-132. 2. Plebani M, et al. Pancreas 2002;24:8-14. 3. Sobrinho LG, et al. Clin Endocrinol (Oxf) 2003;58:686-690. 4. Werner MJ. Chromatogr 1966;25(1):63-70. 5. Kent UM. Methods Mol Bio 1999; 115:11-18.
Conclusions
Protein fractionation using centrifugal ultrafiltration devices is a simple and rapid means of reducing sample complexity and compartmentalize proteins based on molecular weight. Serial enrichment is 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 outperform PES membranes in an enrichment paradigm. Ultrafiltration is a potential alternative to gel filtration chromatography for size based protein enrichment.
Figure 3. Serial enrichment is more efficient than direct enrichment in terms of purity and time
Serial 100 min (150300 L)
M S
100 90 80 70 60 50 40 30 20 10 0 Ultracel PES Device A PES Device B IgG (0.1 mg/mL) IgG (1.0 mg/mL)
100 kDa
Light Chains
10 kDa 10 kDa
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.
Regenerated cellulose membranes (Ultracel membrane) outperform polyethersulfone (PES) membranes used for enrichment. A binary mixture containing Cytochrome c (1 mg/mL) and 155 kDa IgG (0.1 or 1.0 mg/mL) was centrifuged using 100K MWCO devices. Cytochrome c recovery was measured in the permeates. Significantly greater Cytochrome c recovery was observed using Ultracel membrane when the system was challenged with high levels of IgG (1.0 mg/mL).
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Time Required:
Materials
Amicon Ultra-4 device, 4 mL, 10K NMWL Centrifuge with fixed-angle or swinging bucket rotor capable of 3400 x g Kit for microprotein determination (i.e. Sigma cat. no. 610-A/Brilliant blue G/ Coomassie blue) Pipetter with 200 L tip Electrophoresis (agarose gel) and immunofixation equipment with apparatus and reagents
Method
1. Determine the total protein in a 24-hour urine specimen. 2. Fill Amicon Ultra-4 device with 4 mL of urine. 3. Centrifuge at 3400 x g for 3045 minutes (approximately 2550 L concentrate volume). This produces up to a 160-fold increase in concentration. 4. Insert a pipetter into the bottom of the filter unit and withdraw the concentrated sample. 5. Perform agarose electrophoresis on the concentrate 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.
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Acknowledgements
Research using Amicon Ultra devices for urine concentration in this protocol was conducted by Mark Merchant, Ph.D. at Helena Laboratories, Beaumont, TX.
Additional Notes
1. Amicon Ultra devices can also be used to concentrate serum, plasma and cerebrospinal fluid for similar analyses. A concentration of approximately 20 mg/mL is required in order to detect free light chains from diseased patients by agarose electrophoresis. Detection by immunofixation electrophoresis is 10 times more sensitive than by agarose electrophoresis. 2. Normal heterogeneous immunoglobulins may also be seen in urine concentrate with immunofixation electrophoresis. This ladder effect is comprised of microheterogenous light chains. Bence-Jones proteins may be within this ladder. To verify the presence of Bence-Jones proteins requires additional analysis by two-dimensional electrophoresis. 3. If there is excess antigen, dilution of the concentrate will be required until equilibrium is achieved between the antigen (Bence-Jones protein) and the antibody. 4. Millipore also offers static concentrators (Minicon devices) for concentration of Bence-Jones protein in urine.
References
1. Tietz, N. Clinical Guide to Laboratory Tests, 2nd ed. Philadelphia: Saunders, WB; 1990;362363. 2. Kahns L. Clinical Chemistry 1991;37: 15571558. 3. Cleveland Clinic homepage. Accessed July 2002. www.clevelandclinic.org/myeloma/ DiagnosisAndTreatmentOf MultipleMyeloma.html 4. Christenson RH, et al. Clinical Chemistry 1983;29(6):10281030. 5. Christenson RH, and Russell ME. Clinical Chemistry 1985;31(6):973.
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Abstract
This study aims to achieve purification and concentration of a fusion protein composed of the alpha and gamma subunits of the human high affinity IgE receptor. The alpha-gamma fusion protein is purified and then coupled to Sepharose (GE) beads to produce an affinity column for isolating human IgE antibodies.
Method
The alpha and gamma subunit components of the high affinity IgE receptor were transfected into mouse myeloma cell line NS0 and the secreted fusion protein was purified on a Protein G column. The eluted proteins were concentrated from volumes of 5001000 mL to 510 mL using a Centricon Plus-70 centrifugal filter device in a swinging bucket rotor at 3000 x g for 10 minutes (60 mL per filter unit). Buffer exchange was also performed subsequently using the same units in tissue culture grade sodium azide-free PBS. The concentrated samples were then filter-sterilized under sterile conditions using a 0.2 m pore filter.
Figure 1 .
140 120
Results
Spectrophotometric evaluation of alpha-gamma fusion protein recovery using Centricon Plus-70 devices showed a result of 18.8 mg/L of supernatant. HPLC trace analysis revealed the chromatogram shown in Figure 1.
Absorbance Units
100 80 60 40 20 0
0 5 10 15 20 25 30
Acknowledgement
Time (min)
58
Purification of DNA Sequencing Reactions . . . 60 Concentrating and Desalting DNA or RNA . . . 63 Preparing Samples for Forensics ID Analysis . . 66 Concentration of Genomic DNA for Forensic Analysis . . . . . . . . . . . . . . . . . . . . . . 67 Purification of PCR Products . . . . . . . . . . . . . . 68 Quantitative Recoveries of Nanogram Amounts of Nucleic Acids . . . . . . . . . . . . . . . 70 RNA Purification and Preparation of Fluorescent cDNA Probe from Human mRNA . 72 Purification of In Vitro Synthesized mRNA . . . . 76 Effect of Centrifugal Ultrafiltration on Large Fragment DNA Integrity . . . . . . . . . . . . 78 DNA Extraction from Agarose Gels . . . . . . . . 80 PCR Purification . . . . . . . . . . . . . . . . . . . . . . . 82 Enzyme Removal . . . . . . . . . . . . . . . . . . . . . . 84
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Use of Microcon 100 Centrifugal Filter Devices to Purify DNA Sequencing Reactions
Introduction
DNA sequencing reactions performed using ABIs BigDye terminator chemistries require the removal of unincorporated dye-terminators and excess salt prior to injection into a sequencer. The traditional method for purifying DNA sequencing reactions is ethanol precipitationa labor intensive method requiring multiple spins, incubation, and drying that can take up to an hour. Care must also be taken to retain the resulting pellet. Microcon 100 devices can remove contaminating salts and unincorporated dye terminators from DNA sequencing reactions and deliver SEQ quality and read lengths equal to or better than ethanol precipitation in 10 minutes. As capillary-based DNA sequencers become more prevalent, the need for single sample purification of SEQ reactions also increases. The first protocol achieved a time savings of 50 minutes over the traditional ethanol precipitation method while a second protocol, which relies on less vigorous centrifugation, took approximately as long as ethanol precipitation but excelled in experiments in which it was desirable to read close to the primer.
Method
DNA was isolated according to standard protocols and amplified for sequencing analysis on an ABI model 3700 DNA sequencer using polymerase reagents and ABIs BigDye Terminator v. 3.1. Sample was purified using two experimental protocols. Results were assessed using electropherograms, Phred 20 read lengths, and estimation of dye blobs.
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
Equipment
Instrument: ABI sequencer cat. no.3700 Reagent: ABI BigDye Terminator v3.1 Injection parameters: existing parameters developed for Montage Injection solution. Data output: Graphs and electropherograms that show A) removal of dye-terminators (blobs); B) Read length (Phred 20 scores); C) Primer
Results
Key parameters for DNA sequence reactions include quality, read length, sequencing accuracy, ability to read close to the primer, and the presence of dye blobs. The first three factors may be assessed using Phred 20 scoring, which is performed automatically via software. Phred 20 corresponds roughly to a 99% probability that a base call is accurate. Ability to read close to the primer is measured by the Primer + value, which relies on both software and visual verification. Dye blobs are artifacts of unknown origin, occurring during sequencing or cleanup, that may overshadow peaks. Dye blob assessment is typically performed by visually examining the electropherogram.
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Table 1 . Results from a sequence purification using the Microcon 100 device
Dye Terminator Phred 20 Dye Blobs Primer +
1/2X 1/8X
n=48
646 59 656 42
11 10 87
Figure 1 . Electropherogram of a 1/2X SEQ reaction, purified using the Microcon 100 protocol
Phred 20684
Primer + = 8
Dye blobs = 0
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Conclusion
Microcon 100 ultrafiltration devices represent a rapid and efficient alternative to ethanol precipitation when preparing DNA for sequencing analysis. The high-spin protocol outlined above affords a significant reduction (10 minutes vs. 60 minutes) in sample preparation time. For experiments where low Prime + is required, researchers can employ a lowspeed centrifugation protocol that provides read lengths equal to or better than ethanol precipitation, with far less opportunity for user error.
10 1.7 3 2.6 4
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
Phred 20 = 622
Primer + = 1
Dye blobs = 0
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Concentrating and Desalting DNA or RNA with Microcon and Amicon Ultra Centrifugal Filters
Introduction
Centrifugal filter devices serve as powerful tools in molecular biology applications, such as DNA or RNA concentration and desalting procedures. Ultrafiltration (UF) is a pressure-driven, convective process that uses semipermeable membranes to separate species by molecular size and shape. UF is highly efficient, allowing for concentration and desalting at the same time. Unlike the use of chemical precipitation methodologies (i.e., ethanol, phenol/chloroform), there is no phase change, which often denatures labile species. DNA and RNA samples with starting concentrations as low as 5 ng/mL can be routinely concentrated in minutes with 99% recovery of starting material, and without the use of co-precipitants. Centrifugal concentrator devices are ideal for separating high and lowmolecular weight species. Ultrafiltration can also be used to change solvents by diafiltration. In this process, the sample is concentrated, then diluted to the original volume with the desired buffer and concentrated again, thus washing out the original solvent. Millipore ultrafiltration membranes are characterized with well-defined, globular solutes (proteins). Typically, the nominal molecular weight limit (NMWL) for a membrane is the point at which over 90% of a solute with that molecular weight will be retained. To process DNA or RNA, the membrane needs to be characterized according to the number of nucleotides in the fragment. Polynucleotides (DNA and RNA) have tertiary structures that are ordinarily more extended than those of typical globular proteins of similar size. Millipore has determined nucleotide cut-offs (based on the number of bases or base pairs in a fragment of DNA or RNA) that correspond to the NMWL of each of their low binding membranes. The nucleotide cut-off (NCO) indicates the fragment length of single- or double-stranded DNA or RNA that one would expect to recover at 90% efficiency with a unit of the named NCO. It is best to choose the NCO with about half the length of the fragment of interest. For example, selecting a 30K NMWL membrane for a 50 base pair fragment generally results in 90% product recovery. A 10K NMWL membrane would provide for closer to 100% recoveries but would take much longer to process the sample. For nucleic acid samples >500 base pairs, a 100K NMWL membrane is appropriate. Table 1 offers guidelines for DNA/RNA retention based on the nucleotide content of single- and double-stranded pieces. For example, more than 90% of a single-stranded 30-mer will typically be retained by a Microcon 10K NMWL. Concentrating dilute DNA solutions is a key step for many subsequent preparative and analytical procedures. For example, standard plasmid preparations involving cesium chloride, equilibrium centrifugation, and gel filtration yield DNA in large volumes that require concentrating prior to precipitation. DNA concentration is also necessary in purifying restriction fragments from gels.
Table 1 . Nucleotide cut-off guidelines for Microcon centrifugal devices (based on >90% recovery of nucleic acids)
Single-Stranded* Nucleotide Cut-off (bp) Double-Stranded Nucleotide Cut-off (bp)
NMWL
10 30 60 125 300
10 20 50 100 125
*Single-stranded nucleic acids with extensive secondary structure will be better retained than those without.
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There are three major techniques currently available for concentrating nucleic acids: repeated extractions with n-butanol adsorption to ion exchange resin, followed by high salt elution lyophilization The first method has the disadvantage that n-butanol concentrates all solutes, including salt, which tends to co-precipitate with DNA upon addition of ethanol. The second method, ion exchange, aside from requiring buffers of various ionic strengths, yields DNA in a high salt solution. The third method, lyophilization, increases the concentration of buffer components, which can result in degradation of nucleic acids. Ultrafiltration membranes retain DNA or RNA but are permeable by smaller ionic buffer components. Ultrafiltration alone does not change buffer compo-
sition. The salt concentration in a sample concentrated by Microcon centrifugal filter devices will be the same as in the original sample. For desalting, the concentrated sample is diluted with water or buffer to its original volume and spun again in a process called diafiltration. This removes the salt by the concentration factor of the ultrafiltration. For example, if a 500 L sample containing 100 mM salt is concentrated to 25 L (20X concentration factor), 95% of the total salt in the sample will be removed. The salt concentration in the sample will remain at 100 mM. Rediluting the sample to 500 L will bring the salt concentration to 5 mM. Concentrating to 25 L once more will remove 99% of the original total salt. The concentrated sample will now be in 5 mM salt. For more complete salt removal, an additional redilution and spinning cycle will remove 99.9% of the initial salt content (see Table 2).
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Methods
Microcon Device
1. Select a Microcon unit with nucleotide cut-off equal to or smaller than the molecular size of the nucleic acid you want to retain (refer to Table 1). 2. Insert Microcon sample reservoir into one of the two vials provided for each unit. 3. To concentrate (without affecting salt concentration): Pipette up to 500 L of DNA or RNA sample into the reservoir. Spin for recommended time, not exceeding recommended g-force guidelines shown in Table 3. 4. To exchange salt: Add the proper amount of appropriate the diluent to bring the concentrated sample to 500 L. Spin for the recommended time, not exceeding g-force shown in Table 3. To achieve lower salt concentration, repeat the entire step as necessary. NOTE: Do not let filtrate vial overfill. 5. Remove reservoir from vial and invert into a new vial (save filtrate until sample has been analyzed). 6. Spin for 2 minutes at 5001000 x g to recover nucleic acid in the vial. 7. Remove reservoir. Cap vial to store.
1 2 3
88 89 88
Using 1 mL of 25 g/mL E. coli DNA in 6 M CsCl, sample was repeatedly concentrated to 0.1 mL. Sample spun at 2000 x g in a 45 degree fixed angle rotor. The concentrated sample was reconstituted to original 1 mL volume by adding 50 mm Tris. After three spins, CsCl concentration was reduced by four orders of magnitude.
185 50 15 10 25
95 35 8 6 15
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10 15 20 25
98 96 94 96
97 95 94 96
95
95 94 99 100
97
Based on >90% retention. Spinning 50 and 100K devices at higher than recommended g-force may result in lower recovery of DNA.
Table 5 . Recommended g-force and spin time for Amicon Ultra devices
NMWL Maximum G-Force Rating Spin Time (min) at 25 C
40 20 10 20 10
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Preparing Samples for Forensics Identification Analysis Using Microcon Centrifugal Devices
Centrifugal filter devices with ultrafiltration (UF) membranes are used by forensics laboratories to isolate genomic DNA for human identification. The source materials are typically blood stains and/or other bodily fluids obtained from crime scenes, criminal suspects, or human remains. The isolated genomic DNA is used to identify individual persons based on their patterns of Short Tandem Repeats. STRs are polymorphic DNA loci that contain repeated DNA sequences. Because the repeats can vary from two to seven bases in length, many different alleles are possible for each locus. The parallel analysis of 9 to 13 polymorphic STR loci can unequivocally identify an individual. Several suppliers offer STR assay kits. Each supplier offers several versions of their kits with different protocols, modes of operation, and detection systems to suit the users needs. Millipores UF-based centrifugal devices are specified in several of these protocols (AmpFLSTR Profiler, Applied Biosystems; GenePrint STR and GenePrint Fluorescent STR Systems, Promega).
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Figure 1 .
A
Genotype analysis of a semen sample before and after concentration by a Microcon centrifugal filter. Electropherogram A shows a partial profile with a high molecular weight loci in each color missing. Electropherogram B shows a full profile and higher peak heights. Amounts amplified were 0.62 ng for electropherogram A and 1 ng for electropherogram B. The sample was extracted with Chelex beads. The peaks are labeled with allele name, size in base pairs, and fluorescent peak height. The improvement in results is due not only to the increased DNA input but also to the concentration step following extraction.
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Concentration of Genomic DNA for Forensic Analysis Using Amicon Ultra-4 50K Centrifugal Devices
Introduction
Millipores Amicon Ultra-4 centrifugal filter devices provide fast ultrafiltration, with the capability for high concentration factors. The device design incorporates the Millipore Ultracel low binding regenerated cellulose membrane, providing excellent sample recoveries from dilute and complex sample matrices. The Amicon Ultra device is configured in a vertical design, which minimizes solute polarization and subsequent fouling of the membrane. The vertical design and available surface area provide for fast sample processing, high retentate recovery (typically greater than 90% of dilute protein or DNA concentrate), and 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 of genomic DNA from low molecular weight (MW) compounds. The Amicon Ultra device design is compatible with the multiple spin recovery assays used for the purification of genomic DNA for forensic analysis. The concentrate is collected from the filter unit sample reservoir using a pipetter, while the ultrafiltrate can be collected in the provided centrifuge 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.
Amicon Ultra has enabled our testing lab to process samples quicker and with a higher convenience and confidence as the decreased number of handling steps allows us to be free of cross-contamination .
Professor Miguel Paredes, Barcelona Laboratory of Instituto Nacional de Toxicologia y Ciencias Forenses
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Note: The centrifugation time is based on samples containing up to 2 g of genomic DNA. For forensics samples, users will likely need to determine the appropriate centrifugation time as many factors (e.g., DNA concentration and temperature) will affect centrifugation time.
Figure 2 . Effect of MWCO and g-force on the recovery of a 137 bp PCR product using Amicon Ultra-4 100K device
100
% PCR Recovery
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Considerations
For applications involving nucleic acids, strand length is the most useful parameter for selecting the Amicon Ultra device appropriate for a particular application. However other parameters including, DNA concentration, the magnitude of the driving force (g-force) and the salt concentration all act in concert to affect DNA recovery. When purifying PCR reactions for example, optimal yield can be
achieved via dilution of the starting material and running at a relatively low g-force. For PCR fragments, an Amicon Ultra-4 device with a 50K MWCO membrane generally strikes the best balance between speed and recovery. If the DNA sample is in a buffer containing high salt and cannot be diluted, or the device is run at a higher than recommended g-force, a significantly reduced DNA recovery will likely be observed. Under these conditions, higher DNA or RNA recovery can be achieved by using the Amicon Ultra with the next tighter membrane, albeit with a much longer processing time. Note that, for maximal removal of unincorporated single-stranded primers from doublestranded DNA fragments, the molecular weights of the primer and DNA fragment should differ by at least an order of magnitude.
Method
DNA fragments (137, 301, 657 and 1159 bp) were generated by PCR using plasmid DNA as the template. After pooling to minimize deviceto-device variability, 100 L aliquots of the PCR reactions were diluted with 2.0 mL of TE Buffer (10 mM Tris-HCL, pH 8.0, 1 mM EDTA) and added to an Amicon Ultra-4 device with a 50K MWCO membrane. The samples were centrifuged at 2,000 x g for 20 minutes, an additional 2.0 mL of TE buffer was added to the sampled and the PCR products were concentrated by a second 20-minute
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The data presented herein demonstrate the utility of Amicon Ultra-4 devices for the purification of PCR products. By using devices with a 50K MWCO, users can expect to routinely obtain both high recoveries and efficient primer removal that will facilitate downstream applications. Although the analysis performed for this study focused on PCR purification, the general principles are applicable to purification of other nucleic acid preparations.
Removal (% of Challenge)
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spin at 2,000 x g. The purified PCR products were collected by pipette and the recovered DNA quantified by a fluorescent SYBR Green I assay (Molecular Probes). The final sample volume was 87 15 L. The recoveries shown in Figure 1 demonstrate the high recovery that can be obtained from PCR reactions using this approach. The recovery of PCR fragments purified by ultrafiltration can be challenging when the resulting DNA fragments are <1000 bp in length. The amount of DNA recovered after purification can be maximized by using an Amicon Ultra device with a lower MWCO membrane (e.g., 50K versus 100K MWCO) and by centrifuging the devices at 2,000 x g as shown in Figure 2 for a 137 bp PCR fragment. Although increasing the g-force will speed processing time, it will do so at the expense of DNA recovery. For larger DNAs, the effect is less dramatic (data not shown). The quantitative removal of single-stranded DNA primers or oligonucleotides from PCR reactions is critical for downstream applications. In order to demonstrate the ability of Amicon Ultra devices to efficiently remove primers, unpurified PCR products were spiked with 20 pmoles of a DNA reporter primer that had been labeled with fluorescein on its 5 end. This primer (TCAG)5 was designed such that it had minimal secondary structure. After purification of the PCR products with an Amicon Ultra-4 device, the amount of primer remaining in the sample was determined by measuring fluorescence against a standard curve. The average primer removal in this experiment was 97.7 1.4%. This level of primer removal has been shown to be sufficient for DNA sequencing and other important PCR-based applications.
Ultrafiltration-based purification relies on size exclusion to affect the separation of single stranded primers from the double stranded PCR fragments. As discussed above, the major challenge to optimizing ultrafiltration for PCR purification is with small, <1000 bp PCR products. Similarly, the efficiency of primer removal becomes more challenging as the size of the single stranded primer increases. As shown in Figure 4, the ability of an Amicon Ultra device with 30K MWCO membrane to remove primers is significantly diminished as the primer length approaches 50 bases. In contrast, when a 50K MWCO membrane is used, primer removal remains high and is consistent across the range of PCR primers evaluated. Whereas the use of a 100K would be expected to quantitatively remove all but the largest of primers, purification of small (e.g., <300 bp) PCR products is suboptimal when using a 100k MWCO membrane. Therefore, an Amicon Ultra-4 device with a 50K MWCO membrane provides the best balance of primer removal and recovery when used for PCR purification.
Conclusions
Quantitative Recoveries of Nanogram Amounts of Nucleic Acids with Microcon Centrifugal Filters
Introduction
Molecular cloning experiments often require concentration of nanogram quantities of DNA. When dealing with such small amounts of nucleic acids, the use of coprecipitants (tRNA or purified glycogen) is essential for effective DNA recovery1. However, when carrier tRNA cannot be used (for example, when DNA is to be labeled with 32P dATP and polynucleotide kinase), the only method of recovering DNA as a precipitate in ethanol has been the ultracentrifugation method developed by Shapiro2. Ultrafiltration, which has been traditionally used to concentrate and desalt protein samples simultaneously, can be an efficient alternative to ethanol precipitation. In this experiment, three popular ethanol precipitation protocols for DNA and oligonucleotides are compared with ultrafiltration in Microcon centrifugal filter devices. For a detailed discussion on the effects of incubation time, temperature and centrifugation parameters on ethanol precipitation, see Reference 3. were then centrifuged at 12,000 x g in a fixedangle microcentrifuge at 4 C for 15 minutes. The supernatants were carefully removed and pellets resuspended in 50 L of TE buffer. The radioactivity in the precipitates and supernatants was then determined by counting Cherenkov radiation. The percentage of recovered DNA was calculated from precipitates and the initial counts. Each data point in the tables represents the average of at least three samples. To precipitate the oligomer, 60 L of TE buffer containing a given amount of 25-mer and 5 ng of radiolabeled tracer was supplemented with 240 L of 5 M ammonium acetate and 750 L of EtOH. Subsequent steps were identical to those described for DNA.
Ultrafiltration
A solution of DNA (500 L) was placed into a Microcon 30K NMWL unit and spun at 12,000 x g for 10 minutes. A 500 L solution of oligonucleotides in TE buffer was placed into a Microcon 3K NMWL unit and spun for 45 minutes at 12,000 x g. The retentates were recovered by inverting the units and centrifuging at 5001000 x g for 2 minutes. While concentrating the oligomer samples, it is important to avoid high salt concentrations which promote binding of single-stranded nucleic acids to the cellulose-based ultrafiltration membrane. The radioactivity in the retentates and filtrates was determined by counting Cherenkov radiation. Data represent averages of six samples each.
Methods
Plasmid pBR322 was digested with EcoR I and the 3 recessed termini were filled in with alpha 32P dATP, using Klenow fragment of DNA polymerase I. A 25-nucleotide mixed base oligomer was radiolabeled by phosphorylation with bacteriophage T4 polynucleotide kinase.
Ethanol Precipitation
All DNA precipitations were performed in 250 L volume. Each tube contained a given amount of DNA, supplemented with 10 ng of radioactively labeled pBR322 in 0.3 M sodium acetate buffer. To precipitate the DNA, 2.5 volumes of 95% EtOH were added to each tube. The content was well mixed and incubated for the specified period of time at 20 C or 70 C (dry ice). The solutions
Results
Standard methods for ethanol precipitation of nucleic acids and ultrafiltration are compared in Tables 1 and 2. Recovery of DNA was assessed at close to 100%, indicating that no significant amount of nucleic acid was lost to the membrane or device due to adsorption.
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In contrast, DNA recovery using EtOH precipitation varied from as little as 14% as in the case of DNA (10 ng/mL) precipitated for 15 minutes at 70 C to a maximum of 76% after overnight incubation at 20 C.
References
1. Wallace DM. Precipitation of Nucleic Acids. Methods in Enzymology 1987;152:418. 2. Shapiro DJ. Quantitative Ethanol Precipitation of Nanogram Quantities of DNA and RNA. Anal Biochem 1981;110:22931. 3. Crouse J, Amorese D. Ethanol Precipitation: Ammonium Acetate as an Alternative to Sodium Acetate. Focus 1987;9(2):35.
Discussion
Poor recovery of nucleic acids at very low concentration with ethanol precipitation may be partially due to the fact that small amounts of DNA do not adhere well to the tube walls following sedimentation unless high g forces (ultracentrifugation) are employed. The yield of DNA incubated at 70 C is slightly reduced, in agreement with previous studies. Also an overnight precipitation at 20 C can significantly improve recovery. The ultrafiltration devices were found to concentrate and desalt nucleic acids effectively in one step resulting in high recoveries and providing a quick, alternative to ethanol precipitation.
EtOH, 70 C, 15 min EtOH, 20 C, 30 min EtOH, 20 C, 18 hrs Microcon 30K NMWL Device
14 13 31 95
15 20 45 95
23 25 45 98
52 60 76 98
55 72 67 99
EtOH, 70 C, 15 min EtOH, 20 C, 30 min EtOH, 20 C, 18 hrs Microcon 30K NMWL Device
4 6 17 93
4 5 20 94
5 6 15 93
6 4 30 95
33 33 58 95
RNA Purification and Preparation of Fluorescent cDNA Probe from Human mRNA with Microcon Centrifugal Filters
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Introduction
When working with RNA, introduction of RNAse contamination during sample preparation is of major concern. Sample yield and integrity can impact the efficiency of subsequent translations, hybridizations, protein binding, antisense, or ribozyme studies. A series of experiments was performed to determine the effectiveness of using Amicon centrifugal ultrafiltration devices from Millipore to concentrate and diafilter RNA samples. The results indicate remarkably high RNA recoveries and low adsorption losses, especially with prior membrane passivation. Microarrays are efficient tools that enable the high throughput identification of genes that are differentially regulated in response to disease, drugs or other stimuli. With the completion of several key genome sequencing projects, scientists now have the ability to custom design DNA microarrays specific to their research interests. The recent advances in robotics, bioinformatics and detection technologies have greatly simplified the manufacture and analysis of microarrays. However, the successful application of microarray technology requires highly purified, fluorescently labeled cDNA probes. The application of ultrafiltration technology to this challenge has resulted in a robust, efficient and rapid method for the genera-
tion of high quality fluorescently labeled probes suitable for use with microarrays. The protocol below describes a method for the generation and purification of fluorescently labeled probes using a Microcon 30K NMWL centrifugal filter device.
Figure 1 .
Concentration of RNA transcript. RNA is intact and recovered with high efficiency. Lane 1: Starting material Lane 2: RNA concentrated in a Microcon 100K NMWL device Lane 3: RNA concentrated in a Microcon 30K NMWL device
1.4 kb
0.68 kb
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Method
Generation and Purification of Fluorescently Labeled Probes
1. To anneal primer, mix 2 g of mRNA or 50100 g total RNA with 4 g of a regular or anchored oligo-dT primer in a total volume of 15.4 L as shown in Table 1. 2. Heat to 65 C for 10 minutes and cool on ice. 3. Add 14.6 L of reaction mixture each to Cy3 and Cy5 reactions as shown in Table 2. 4. Incubate at 42 C for 1 hour. 5. Add 1 L SSII (RT booster) to each sample. Incubate for an additional 0.5 1 hours. 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.
7. Neutralize by addition of 15 L of 0.1 N HCl. 8. Add 380 L of TE (10 mM Tris, 1 mM EDTA) to a Microcon 30K NMWL device column. Next add the 60 L of Cy5 probe and the 60 L of Cy3 probe to the same Microcon device. NOTE: If re-purification of Cy dye flow-through is desired, do not combine probes until Wash 2. 9. Wash 1: Spin column for 7 to 8 minutes at 14,000 x g. 10. Wash 2: Remove flow-through and add 450 L TE and spin for 78 minutes at 14,000 x g. It is a good idea to save the flowthrough for each set of reactions in a separate microcentrifuge tube.
x 1 to 15.4 15 .4
y 1 to 15.4 15 .4
5X first-strand buffer* 0.1 M DTT Unlabeled dNTPs Cy3 or Cy5 (1 mM, GE Healthcare) Superscript II (200 U/L, Gibco-BRL) Total volume
dATP (100 mM) dCTP (100 mM) dGTP (100 mM) dTTP (100 mM) Purified H2O Total volume
25 25 25 10 15 100
25 25 25 10
*5X first-strand buffer: 250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2
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11. Wash 3: Remove flow-through and add 450 L 1X TE, 20 g of Cot1 human DNA (20 g/L, Gibco-BRL), 20 g polyA RNA (10 g/L, Sigma cat. no. P9403) and 20 g tRNA (10 g/L, Gibco-BRL cat. no. 15401-011). Spin 710 minutes at 14,000 x g. Look for concentration of the probe in the Microcon device. The probe usually has a purple color at this point. Concentrate to a volume of less than or equal to the volume listed in the Probe and TE column in Table 3. These low volumes are attained after the center of the membrane is dry and the probe forms a ring of liquid at the edges of the membrane. Do not dry the membrane completely. 12. Invert the Microcon device into a clean tube and spin briefly at 14,000 RPM to recover the probe. 13. Select the appropriate row from Table 3. Adjust the probe volume to the value indicated in the Probe and TE column. 14. For final probe preparation add 4.25 L 20X SSC and 0.75 L 10% SDS. When adding the SDS, be sure to wipe the pipette tip with clean, gloved fingers to remove excess SDS. Avoid introducing bubbles and never vortex after adding SDS. The probe is ready for hybridization.
Results
RNA recovery is a function of the initial RNA concentration and the buffer salt concentration. Figure 2 shows RNA transcript recovery in Microcon 100K. NMWL unit as a function of RNA concentration. For all concentrations evaluated, retentate recovery is above 85%, even at an initial concentration as low as 25 ng/mL. Figure 2 also displays the material monitored in the filtrate and on the membranes of the Microcon units. (Membranes were removed and counted without further treatment.) The amount of RNA in the filtrate does not vary significantly as a function of concentration. The RNA recovered on the membranes varies from <1% at high initial concentrations to 4% at the lowest concentration tested. Millipores Amicon centrifugal ultrafiltration devices contain low-binding cellulosic Ultracel-YM membranes. Nitrocellulose is commonly used to immobilize RNA, generally in high salt concentrations. Figure 3 shows RNA recovery as a function of buffer salt concentration. The data show that both retentate and total recoveries fall as salt concentration increases. An increase in the amount of material on the membrane is also seen (from 2% to 6%) as the salt concentration increases from 10 to 500 mM. Similar trends are seen in the use of Microcon
22 x 22 22 x 40 22 x 60
15 25 35
12 20 28
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0.5
1.0
5.0
10.0
Microcon Filtrate
Microcon Retentate
Conclusion
The results shown here demonstrate that RNA samples can be concentrated or diafiltered with Microcon concentrators without loss of RNA integrity. RNA recovery in the retentate is typically >85%. As the salt concentration of the buffer system increases, RNA retentate recovery decreases, principally due to RNA adhering to the device rather than to the membrane. RNA recovery from solutions with high initial salt concentrations can be significantly improved by pre-treating the devices with a 5% SDS solution.
RNA recovery in Microcon concentrators. RNA was concentrated as described in text. Cherenkov counts of Microcon retentate, filtrate and membrane (n=3 units) were compared to total starting counts.
Figure 3 .
100% 80%
Acknowledgements
Fluorescent probe preparation protocol courtesy of Patrick Brown, Max Diehn, and Ash Alizadeh, Stanford University School of Medicine.
100
200
500
Retentate
Improvement in RNA recoveries using Microcon devices (n=6) which were pre-treated with a 5% SDS solution.
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100K NMWL devices, except that retentate recoveries fall to 60% at 500 mM salt. It was possible to count the devices themselves in addition to the filtrate and membrane. The results show a slight increase in counts on the membrane with increasing salt concentration. Significantly more (from 9% to 17%) material remained on the device as salt concentration increased. Passivation increased RNA recovery to 80%, regardless of initial salt concentration. This is attributed to a decrease in the amount of RNA adhering to the device. When the devices were passivated, RNA loss due to adsorption was reduced to 2%, regardless of salt concentration.
Figure 2 .
100% 80%
60% 40%
Methods
RNA Transcription
For our studies we chose plasmid pGEM-luc containing the luciferase gene (luc) in the center of a multiple cloning cassette of the pGEM-11Zf (-) plasmid (Promega). DNA template was linearized with XhoI, followed by enzyme and salt removal by diafiltration in Microcon 100K NMWL devices. Linearized template was transcribed, using MEGAscript kit (Ambion) according to the recommended protocol. After the reaction was completed (3 to 4 hours), template DNA was degraded with DNase I and the reaction mix added to a Microcon 30K NMWL device filled with 450 L of water. The device was spun for 20 minutes at 12,000 x g in a temperature-controlled centrifuge at 4 C. Purified, concentrated RNA was recovered by inverted spin. For the preparation of capped transcript, cap analog m7G (5) ppp (5) G (New England Biolabs, Inc.) was included in the transcription reaction and the level of GTP reduced (4:1 ratio of cap analog to GTP). To purify the transcript by phenol/chloroform extraction, the reaction mix was diluted with water and a one-tenth volume of ammonium acetate stop solution was added. The mixture was extracted once with phenol/chloroform, followed by chloroform extraction. RNA was precipitated with isopropanol and the pellet resuspended in distilled water. Alternatively, LiCl precipitation solution (one-half volume) was added to the reaction mix, followed by incubation at minus 20 C for 1 hour. RNA was pelleted by centrifugation and dissolved in water. Size and integrity of the in vitro transcription products were assessed by running an aliquot of the purified RNA transcript on a formaldehyde/formamide agarose gel. Ethidium bromide was added to the RNA before lading on the gel to stain the RNA sample and keep background fluorescence low2.
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Translation In Vitro
In vitro translations were performed in the Flexi Rabbit Reticulate Lysate System (Promega) according to standard luciferase RNA translation conditions with minor modifications (Rnasin Ribonuclease inhibitor was omitted and 35S-methionine added). Results of translation were analyzed by determination of percent incorporation of 35S-methionine and fold stimulation, compared to controls without RNA. Minimum acceptable stimulation was 8-fold.
References
1. Krowczynska AM. BioSolutions 1993;2(1):12. 2. Ogretmen B, Ratajczak H, Kats A, Stark BC. Biotechniques 1993;14(6):932-5. 3. Polayes D. Focus 1991;13(4):1302. 4. Dasso MC, Jackson RJ. Nucl Acid Res 1989; 17:3129.
Figure 1 .
Comparison of pGem-luc transcript purification methods. Transcripts were synthesized in 20 L reactions. After DNase I treatment, RNA was purified from the reaction mix. 500 ng of purified RNA were run on 1% agarose/ formaldehyde gel. Lane 1: 0.161.77 kb RNA ladder (Gibco BRL) Lane 2: RNA purified in Microcon 30K NMWL device Lane 3: RNA purified by phenol extraction Lane 4: RNA purified by LiCl precipitation
Results
Aliquots of RNA transcript purified by different methods (ultrafiltration, phenol extraction and LiCl precipitation) were run on a denaturing agarose/ formaldehyde gel. Results are shown in Figure 1. The banding pattern of the 1.7 kb RNA transcripts is identical regardless of purification method. Similar results were obtained in the case of capped transcript (results not shown). 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 uncapped mRNA (Figure 2). At higher mRNA levels, the translation of both transcripts was comparable. Similar behavior was observed with CAT mRNA3. Even relatively high levels of mRNA did not cause the decrease in translational efficiencies noted by other groups4. This result could be attributed in part to the lack of inhibitory contaminants in the mRNA preparation. We also checked the effect of the RNA clean-up method on in vitro translational efficiency. For details of various procedures, see the Methods section. RNA purified by each of the methods (1 g) was translated and analyzed. While there were no observable differences between these RNAs by gel electrophoresis analysis (Figure 1), RNA purified by ultrafiltration gave twice the translation efficiencies of phenol-extracted RNA.
Figure 2 .
140 120 100
80 60 40 20 0
0 0.5 1.0 1.5 2.0
g RNA/50 L Reaction Effect of pGem-luc RNA concentration on in vitro translation. Increasing amounts of uncapped Luc RNA transcripts and capped Luc RNA were used in translation reactions. Incorporation of 35S-methionine was determined by TCA precipitation. Both RNA transcripts were purified with Microcon 30K NMWL devices.
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cpm x 104
Methods
DNA Ladder
As a model system to check for the introduction of single-strand nicks during the ultrafiltration spin, supercoiled Ladder DNA (Gibco-BRL, Gaithersburg, MD) which ranges in size from 2 to 8 kb was used. DNA diluted with TE buffer was loaded into Microcon 30K NMWL devices. The Microcon units were spun for 7 minutes at 12,000 x g. The concentrated DNA sample (retentate) was diluted
Figure 1 .
Supercoiled DNA Ladder. Lane 1: Starting material Lanes 2, 3: Retentate from DNA ladder spun 3 times at 12,000 x g in Microcon 30K NMWL device
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The results are shown in Figure 2. As in the case of the DNA Ladder, the concentrated samples (lanes 2, 4 and 6) appear to be identical with their corresponding starting material. Again, there is no evidence of conversion of the supercoiled form to the relaxed form after exposure to g forces of 5,000 x g for 90 minutes and 12,000 x g for 21 minutes during the concentration spins.
Figure 2 .
Specific plasmid DNA Lane 1: pBR322 starting material Lane 2: pBR322 spun in Microcon 30K NMWL device Lane 3: pSPT18 starting material Lane 4: pSPT18 spun in Microcon 30K NMWL device Lane 5: pXT1 starting material Lane 6: pXT1 spun in Microcon 30K NMWL device
Figure 3 .
Large fragment DNA Lane 1: Lambda DNA starting material Lane 2: Lambda DNA spun in Microcon 30K NMWL device Lane 3: BacPAK DNA starting material Lane 4: BacPAK DNA spun in Microcon 30K NMWL device
Conclusions
DNA samples of up to 49 kb were concentrated repeatedly without any loss of sample integrity. Some loss of integrity was observed with a 125 kb sample, although it was not complete and represents a small percentage of the total DNA in the sample. For large fragments of DNA, centrifugal ultrafiltration provides a fast and efficient method to concentrate or desalt the sample. It results in high recovery of intact product.
References
1. Takagi S, Kimura M, Katsuki M. BioTechniques 1993;14(2):21821. 2. PCR is covered by U.S. patents issued to Hoffmann-LaRoche, Inc. 3. Sheng N, Zhang J, Whitton JL, McKee T. BioTechniques 1993;14(5):7814. 4. Ruano G, Pagliaro EM, Schwartz TR, Lamy K, Messina D, Gaensslen RE, Lee HC. BioTechniques 1992;13(2):26674.
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DNA Extraction from Agarose Gels with Montage Gel Extraction Kit or Ultrafree-DA Centrifugal Filters
Introduction
The Ultrafree-DA device is designed to recover 100 to 10,000 bp DNA from agarose gel slices in one 10-minute spin. It consists of a pre-assembled sample filter cup with an agarose gel nebulizer and a microcentrifuge vial. The device uses gel compression to extract DNA from the agarose. Centrifugal force collapses the gel structure, drives the agarose through a small orifice in the gel nebulizer and captures the resultant gel slurry in the sample filter cup. As the agarose is compressed at 5,000 x g, DNA is extruded from the gels pores. The gel matrix is retained by the microporous membrane, and the DNA passes freely through the membrane. DNA can then be recovered in the filtrate vial. The Montage Gel Extraction Kit consists of 50 Ultrafree-DA centrifugal filters as well as a modified TAE buffer that allows the casting and running of the gel from which the DNA fragment is to be extracted. DNA prepared with the Ultrafree-DA centrifugal filter requires no further purification for most applications, including cloning and radioisotopic or fluorescent DNA sequencing. Since agarose gel electrophoresis has high resolving power, the small and large non-specific amplification products that frequently interfere with cloning and sequencing after PCR (polymerase chain reaction) are completely removed from the product.
Materials
Microcentrifuge Pre-assembled Ultrafree-DA centrifugal filter device or Montage Gel Extraction Kit Modified TAE* electrophoresis buffer (40 mM Tris-acetate, pH 8.0, 0.1 mM Na2EDTA) SeaKem agarose (FMC BioProducts) or equivalent Long-wavelength UV lamp Scalpel or razor blade
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 reactions 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 downstream enzymatic treatments, many of which are dependent on magnesium.
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Procedure
1. Electrophorese 30 L of PCR product or other DNA through a <1.25% ordinary agarose gel, prepared in modified TAE buffer with ethidium bromide (0.5 g/mL). 2. Locate the band of interest with a long wavelength UV lamp or transilluminator. With a razor blade or scalpel, cut out the slice of agarose (<100 L or 100 mg) containing the band of interest. Trim any away excess agarose from band. 3. Place the gel slice into the gel nebulizer/sample filter cup/filtrate vial assembly and seal the device with the cap attached to vial.
4. Spin at 5,000 x g for 10 minutes. Centrifugation forces the agarose through the gel nebulizer, converting it to a fine slurry that is captured by the sample filter cup. Extruded DNA in electrophoresis buffer passes through the microporous membrane in the sample filter cup and collects in the filtrate vial. 5. DNA in the filtrate is now ready for sequencing or cloning without further purification. Discard the gel nebulizer and sample filter cup and store the DNA in the capped filtrate vial.
Results
Gel compression is a quick and easy technique for recovering DNA from an agarose gel slice.
Table 1 . Effect of gel disruption on typical DNA recoveries from agarose gels
% DNA Recovered from Intact Gel %DNA Recovered from Gel Disrupted by Gel Nebulizer
39 43 55 * 14 * *
ND 71 77 47 35 32 29
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100
74
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Figure 1 .
Primer DNA Salt dNTP
Step 1
Step 2
Step 3
Materials
Variable speed microcentrifuge Montage PCR filter unit Purified water (such as Milli-Q water) or TE buffer PCR reaction (aqueous phase)
Results
Montage PCR filter units were used to purify 100 L PCR reactions according to the specified protocol. After recovering the DNA fragments (n = 10) using a reverse spin, the samples were separated by agarose gel electrophoresis. Recoveries of the various PCR products were determined by densitometry (Figure 1). The Montage PCR filter unit is a convenient method for single-sample PCR purification. The high-performance device purifies PCR products in a single centrifugation step. Purified samples are ready for downstream applications with no additional purification steps.
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
Method
1. Insert the Montage PCR sample reservoir into one of the two vials provided. 2. Fill the reservoir with 300 L purified water or TE buffer. Add 100 L PCR reaction to the reservoir (step 1). Smaller volumes of PCR product may be used, but the volume should be adjusted to a final volume of 400 L. 3. Spin the Montage PCR device at 1000 x g for 15 minutes (step 2). NOTE: For optimal recovery, do not centrifuge longer than the specified 15 minutes or greater than 1000 x g. 4. To recover DNA, remove the sample reservoir from filtrate collection vial and place in a clean vial. 5. Add 20 L purified water or TE buffer to sample reservoir. 6. Invert the reservoir and spin at 1000 x g for 2 minutes to retrieve the purified PCR (step 3).
References
1. PCR is covered by U.S. patents issued to Hoffmann-LaRoche, Inc.
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Figure 2 .
100% 80%
Percent Recovery
Figure 3 .
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.
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Method
In order to ensure that each enzyme assay was detecting trace amounts of restriction enzymes in Micropure-EZ filtrates, serial dilutions of Bgl I (representing 2 units, 0.4 units, 0.08 units, and 0.016 units of total activity in 10 L) were carried out in a reaction mix* prepared with either sterile DI water (sdH2O) or with sdH2O that had been filtered through Micropure-EZ devices and then incubated. Comparison of the resultant restriction digests after agarose gel electrophoreses verified that aqueous extractables from Micropure-EZ devices did not inhibit the enzyme. Also, these standard curves determined the sensitivity of the enzyme assay (i.e., the theoretical amount of residual enzyme activity that could be detected, if present). To stress test Micropure-EZ devices sufficiently with enzymes and DNA, several extreme operating conditions were used consistently. Micropure-EZ devices were challenged with 50 units of Bgl I in the presence of decoy DNA (1 g of pBR322) and 5 g of bovine serum albumin (BSA). Devices were spun at 14,000 x g for 30 seconds. The filtrates were then assayed for residual enzyme activity by adding 1 L of pUC19 DNA and 0.5 L of 100X BSA to the filtrate, mixing thoroughly and incubating
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 1 .
DNA Enzyme Salt Micropure-EZ
Remove Enzyme Restriction and other enzymes are absorbed by Micropure-EZ. dsDNA passes freely. Concentrate DNA DNA is retained by ultrafilter in Microcon. Salts pass freely.
Micropure-EZ
Remove Enzyme Restriction and other enzymes are absorbed by Micropure-EZ. dsDNA passes freely.
Microcon Assembly
30 seconds
3 minutes
*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)
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at 37 C for 1 hour. After a brief centrifugation to collect the condensate at the bottom of the microcentrifuge tubes, 1 L of 0.5 M disodium EDTA and 10 L of 5X loading buffer were added to stop the reaction. The negative control was a portion of DNA master mix. Control digests of pBR322 DNA and pUC19 DNA were carried out separately.
which was added directly to the filtrate and incubated, appeared completely intact (lanes 1114). Since the standard curve indicated that 0.08 units would be detected if it were present, we calculated that at least 99.8% of Bgl I was removed and/or inactivated. Removal, rather than inactivation, is the probable mechanism by which Micropure-EZ devices operate, since in separate experiments we were unable to detect any enzyme (irrespective of activity) in Micropure-EZ filtrates using very sensitive HPLC methods (data not shown).
Summary
Enzyme removal and excellent DNA recovery are accomplished with Micropure-EZ devices in a single 60-second spin without any pre-wetting, binding, washing or elution step. Cumbersome phenol/ chloroform extraction and the associated hazardous waste accumulation are avoided. An additional line of evidence for the suitability of Micropure-EZ devices in molecular biology applications is that restriction digest purified with Micropure-EZ devices alone are readily ligated and cloned into plasmid DNA (M.H.A.L., unpublished observations). Micropure-EZ devices used alone or with Microcon devices permit rapid and efficient sequential enzymatic DNA manipulations.
Figure 2 .
Analytical agarose gel, demonstrating the detection limits for Bgl I and Bgl I removal by Micropure-EZ devices. Lanes 14: Lane 5: Lanes 69: Lane 10: 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 Undigested DNA master mix used as substrate for the standard curve containing pUC19 and pBR322 DNA 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 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: Lane 16: Lane 17: Lane 18: Uncut pBR322 BgI Icut pBR322 Uncut pUC19 BgI Icut pUC19
10
11
12
13
14
15
16
17
18
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AMV reverse transcriptase Calf intestinal alkaline phosphatase DNase I (bovine pancreas) Exonuclease III (E. coli) MMLV reverse transcriptase Mung bean nuclease Proteinase K (Amresco) T4 DNA ligase T4 DNA polymerase Taq DNA polymerase Terminal deoxynucleotidyl transferase Acc I Apa I BamH I Bcl I
Bgl I BsiW I BssH II BstN I Dpn I EcoR I Hae III Hinc II Hind III Hpa I Kpn I Mbo I Mlu I Nco I Nde I NgoM I Nhe I Not I
T4 polynucleotide kinase** 50 U
In tests, all enzymes were removed from solutions containing DNA or RNA (in the case of RNase A). Five g of bovine serum albumin were also present during removal of restriction enzymes. Removal was indicated by undetectable or inconsequential enzyme activity in filtrate (<0.08% residual in the case of T4 polynucleotide kinase).
*New England Biolabs unit definition **T4 polynucelotide kinase is not recommended with oligonucleotides (dsDNA only). The results suggest that this kinase mediates the binding of oligos to the membrane in Micropure-EZ device, causing oligo loss.
Table 2 . Enzymes not removed by Micropure-EZ devices P ro T o C o l s f rNu cl eli c iCciaCsid s E n z y m e Re m ova l o nUC e A d
Enzyme Challenge Enzyme Challenge
Bacterial alkaline phosphatase DNA polymerase I (Klenow) Exonuclease I Pfu DNA polymerase Vent DNA polymerase Shrimp alkaline phosphatase RNase A (bovine pancreas) ApaL I Bgl II BsoB I Cla I
0.6 U 20 U 50 U 2.5 U 4U 1U 1 g 10 U 50 U 50 U 25 U
Eae I EcoR V Hinf I Msp I Pvu I Pvu II Sau3A I Sfi I Sma I Xba I
15 U 50 U 50 U 100 U 25 U 50 U 20 U 50 U 25 U 50 U
In tests, Micropure-EZ devices did not remove the indicated number of units of the listed enzymes. It may be effective in removing a lower number of units.
*T4 RNA ligase is not recommended. Results suggest this ligase mediates binding of nucleic acids to the membrane in Micropure-EZ devices, causing sample loss.
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P ro T o C o l s f o r v irU s C o nC e n T r aT i o n 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
Method
Phi-6 production
Note: All manipulations should be performed using aseptic technique in a laminar flow hood. 1. Grow fresh culture of Pseudomonas pseudoalcaligenes until it reaches the desired optical density of 0.1 absorbance units at 600 nm. 2. Inoculate the culture with enough phi-6 to achieve a multiplicity of infection (MOI) of 0.5. 3. Return the phi-6 containing Pseudomonas pseudoalcaligenes culture to the platform shaker and incubate at 26 C, 125 RPM, for 1824 hours. 4. After the overnight incubation, aliquot the culture into four 250 mL centrifuge bottles. 5. Centrifuge the bottles for 30 minutes at 16,000 x g. 6. Remove the supernatant and transfer to a sterile container.
density gradient centrifugation, followed by extensive dialysis3,4. Phage titers are then determined by plaque assays. For some experiments, high titer bacteriophage stock is desired. Traditionally, phage particles were concentrated by polyeteylene glycol (PEG) and dextran sulfate precipitation, precipitation by acid, or differential or equilibrium centrifugation5. Recently, different modifications of the Sambrook method are being used6. Phage is concentrated by precipitation with sodium chloride and PEG and resuspended to the desired titer. The process requires 612
hours of incubation with PEG and a lengthy pellet resuspension process to achieve high particle recovery 7,8.
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Concentration using Amicon Ultra-4 or Ultra-15 devices with 50 kDa NMWL membrane
1. Clarify phi-6 supernatant using a Stericup filter unit (Millipore cat. no. SCGP U05 RE), a Steriflip filter unit (Millipore cat. no. SCGP 005 25), or a Millex-GP syringe filter (Millipore cat. no. SLGP 033 RB) with lowbinding 0.22 m PES membrane. 2. Load appropriate volume of clarified phi-6 supernatant (4 or 15 mL) into an Amicon Ultra-4 device (Millipore cat. no. UFC8 050 24) or an Amicon Ultra-15 device (Millipore cat. no. UFC9 050 24) containing 50,000 NMWL regenerated cellulose membrane. 3. Centrifuge the device (4 or 15 mL) for 15 minutes at 1500 g. 4. The recoverable volume should be 80100 L for Amicon Ultra-4 devices and 350500 L for Amicon Ultra-15 devices. Note: Avoid concentrating samples below recommended volumes as this may lead to decreased phage recovery.
6. Set retentate pressure by slowly adjusting retentate valve clockwise until retentate pressure is 10 psi. 7. Adjust pump speed and retentate valve restriction to achieve desired feed pressure of 30 psi and retentate pressure of 10 psi. Note: Do not exceed feed pressure of 60 psi. 8. Filter the solution until the desired concentration or volume is obtained. 9. Stop pump. 10. Disconnect pump outlet tubing from pump outlet port and place in product recovery collection vessel. 11. Disconnect retentate tubing from retentate in port. Fluid should now drain by gravity. If additional drainage is required, a syringe can be placed on the end of the retentate tube and fluid can be blown down. 12. Replace retentate tubing in retentate port. Reconnect pump outlet tubing. 13. Disconnect FEED IN tubing and place in collection vessel. Open tank outlet valve, turn pump speed up to drain reservoir. 14. Reconnect the pump outlet tubing to the FEED IN port.
Concentration using Pellicon XL50 device with PLCTK membrane and Millipores Labscale TFF system
Note: The Labscale TFF system (Millipore cat. no. XX42 LSS 13) is not required to use Pellicon XL50. A peristaltic pump may alternatively be used (see Millipore user guide P60085). 1. Perform flush, integrity, and permeability tests on Pellicon XL50 device with PLCTK membrane (Millipore cat. no. PXCO 30C 50) as indicated in the Pellicon XL50 User Guide. 2. Remove reservoir cover and load up to 500 mL phi-6 supernatant into reservoir of Labscale TFF system. 3. Ensure vent port is open by removing plug from VENT port and leaving open or installing a Millex filter. 4. Open tank outlet valve. 5. Turn on pump and increase speed until feed pressure reaches 20 psi. Note: Check system connections for leaks. Tighten connections as required.
Results
Millipore ultrafiltration devices with Ultracel regenerated cellulose membrane offer low non-specific binding and high retentate recovery. The vertical membrane reduces concentration polarization for ultra-fast concentration times and is especially beneficial for turbid and viscous solutions. As shown in the protocol above, phage titer can be increased up to 30-fold in 15 minutes by centrifugal ultrafiltration for under 15 mL volumes. Amicon Ultra-4 and Ultra-15 devices with 50 kDa NMWL membranes can be used successfully to concentrate the phage phi-6 out of bacterial culture supernatant. The same devices with 100 kDa NMWL membranes are not recommended because they lead to a low virus recovery and allow virus to break through into the filtrate.
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For large volume concentration, tangential flow filtration (TFF) should be used. Table 1 shows phage concentration results using a Pellicon XL50 cassette with the Labscale TFF system. The Pellicon XL50 cassette couples Millipores ultrafiltration membranes with the linearly scalable TFF cassette for processing 501000 mL volumes. The Ultracel membrane with 30 kDa NMWL is recommended. Twenty-fold concentration of phage solution is usually achieved in 3050 minutes.
5. Bachrach U, Friedmann A. Appl Microbiol 1971;22(4): 706-715. 6. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1989 7. Bahns JT, Liu CM, Chen L. Protein Science 2004;13:2578-2587. 8. Gill JJ, et al. Chemother 2006;50(9): 2912-2918. 9. Meile L, Abendschein P, Leisinger T. J Bacteriol 1990;172(6):3507-3508. 10. Middelboe M, et al. Aquat Microb Ecol 2003;33:1-10. 11. Rembhotkar GW, Khatri GS. Anal Biochem 1989;176(2):373-4. 12. Alonso MC, Rodriguez J, Borrego J. J Internatl Microbiol 1999;2:227-232.
References
1. Pennazio S. Riv Biol 2006;99(1):103-29.
P ro T o C o l s f o r v irU s C o nC e n T r aT i o n 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
2. Fischetti VA, Nelson D, Schuch R. Nature Biotechnology 2006;24:1508-1511. 3. Hansen MR, Mueller L, Pardi A. Nature Structural Biology 1998;5:1065-1074. 4. Zhilenkov EL, et al. Virology Journal 2006;3:50-55.
Initial volume (mL) Initial viral titer, pFU/mL Final volume (mL) Final viral titer, pFU/mL Concentration factor Phage recovery (%)* Phage in the filtrate, pFU/mL
*We consistently observe higher than 100% recovery after phage concentration. This can be explained by increased infectivity of phage particles at higher concentrations and agrees with other published data12.
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Method
1. Packaging cells: Host cells are plated to achieve 8085% confluence 2. Transfection: Cells are either transfected with recombinant plasmid(s), or infected with viable virus particles. 3. Harvesting virus: Depending on the virus, either cell supernatant or pelleted cells, or both, are used to purify the recombinant virions. Three to four rounds of freeze/thaw cycles are usually applied to release virus from cells. Cell debris is then separated from cell lysate by centrifugation. 4. Virus purification: Virus is purified from cell lysate by density gradient ultracentrifugation or column/membrane chromatography. In some cases, affinity chromatography with heparin or mucin sepharose13,14,15 can be used.
For virus propagation, HEK-293T cells were transfected with helper plasmids and lentiviral plasmid expressing copGFP (System Bioscience). 48 hours after transfection viral supernatant was collected and 10 mL was concentrated using Amicon Ultra-15 100 kDa MWCO membrane to 200 L. 50 L of concentrated viral stock was used for infection of 10E6 cells.
Data courtesy of Drs. Leila Aminova and Ambreena Siddiq, Neuroprotection Laboratory of Beth Israel Medical Center.
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Purified virus solutions obtained via these methods are then buffer exchanged using either passive dialysis or diafiltration into an appropriate storage solution.
Virus Concentration
In order to achieve a high titer of viral stock, the purified virus can be concentrated using centrifugal ultrafiltration. The correct choice of device, membrane material, MWCO, centrifugation speed, centrifugation time and buffer composition are critical for high recovery of infective viral particles. Amicon Ultra centrifugal ultrafiltration devices with a 50 KDa NMWL Ultracel regenerated cellulose membrane have been demonstrated to successfully concentrate adenovirus and AAV solutions. For lentivirus concentration, devices with both 50 and 100 kDa membranes can be used.
P ro T o C o l s f o r v irU s C o nC e n T r aT i o n C o n ce n t r a t io n of A n i m a l V i ru s e s U s i n g U l t r af i l t ra t i o n
Table 1 shows typical results of concentration of adenovirus, lentivirus and AAV from both crude cell lysate and virus purified by chromatography. It is important to remember that most of the viruses cannot be efficiently concentrated and stored in low salt buffers. Centrifugal concentration of the virus in <200 mM salt solutions will lead to virus aggregation and decreased infectivity. An example of a recommended storage solution is 20 mM Tris, pH 8.0, 250 mM NaCl, 10 mM MgCl2, 5% sorbitol and 0.001% PF68 (pluronic acid)16. Concentrated viral stock can also be filter sterilized through 0.22 m filters. For example, Steriflip-GP filter units (cat. no. SCGP 005 25) can be used for volumes of 50 mL or less or Ultrafree-MC GV (cat. no. UFC3 0GV 0S) can be used for volumes of <0.5 mL.
Adenovirus in crude cell lysate Adenovirus purified, in 1M salt buffer AAV in crude cell lysate
4 4 4 10
20 10 35 45
>100 85 50 76
Amicon Ultra-15, AAV in crude 50 kDa MW cell lysate, diluted in 100 mM Tris, 200 mM NaCl buffer Amicon Ultra-4, AAV in crude 50 kDa MW cell lysate, diluted in 100 mM Tris, 200 mM NaCl buffer Lentivirus in cell culture supernatant Lentivirus in cell culture supernatant Lentivirus in cell culture supernatant Amicon Ultra-4, 100 kDa MW Amicon Ultra-4, 50 kDa MW Centricon 70 Plus, 100 KDa MW
0.5 x 107
200
20
100
20X
4 4 65
40 40 800
30 30 30
>100 93 81
*We do not recommend concentrating the virus to higher than 1013 particles/mL due to potential virus aggregation17.
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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.
9. Elahi SM, et al. Gene Ther 2002;9:1238. 10. Choi VW, et al. Curr Gene Ther 2005; 5:299. 11. Blanche F, et al. Gene Therapy 2000;7: 1055-1062. 12. Shabram PW, et al. Hum Gene Therapy 1997; 9:453-465. 13. Clark KR, et al. Hum Gene Therapy 1999; 10:1031-1039. 14. Zolotukhin S, et al. Gene Therapy 1999;6: 973-985. 15. Auricchio A, et al. Hum Gene Therapy 2001; 12:71-76. 16. Alejandra E, Arbetman, AE, et al. Journal of Virology 2005;79(24):15238-15245. 17. Galdiero F. Arch Virol. 1979;59(1-2):99-105.
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a P P e nd i x H i g h T h ro u g h p u t A p p l i c a t io n s
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Glossary
Asymmetric Membrane1 Membrane constituted of two or more structural planes of non-identical morphologies. Batch Process A fixed volume of solution contained in a tank to which the concentrate is returned during the process. Composite Membrane1 Membrane having chemically or structurally distinct layers. Concentration Polarization Accumulation of rejected solute on the membrane surface. Depends on interactions of pressure, viscosity, crossflow (tangential) velocity, fluid flow conditions, flow channel conditions and temperatures. Crossflow (Tangential Flow) Solution flows across (tangential to) a membrane surface. Facilitates back diffusion of solute from that surface into the bulk solution, counteracting concentration polarization. Diafiltration Removal of small components from the retained species during ultrafiltration by adding water or buffer solution to the retentate. See page 7 for further discussion. Dialysis Diffusive transport of ions or other small molecules through a membrane barrier that contacts solvent on both sides. Downstream1 Side of a membrane from which the permeate emerges. Feed (Sample) The starting solution (sometimes the solution remaining upstream of the membrane). Fluid Velocity The flow rate of solution across the membrane surface in cross (tangential) flow. Related to hydraulic pressure drop. Flux The filtration rate through the membrane per unit area. Fouling Irreversible decline in membrane flux due to deposition and accumulation of submicron particles and solutes on the membrane surface. Also, crystallization and precipitation of small solutes on the surface and in the pores of the membrane. Not to be confused with concentration polarization. Membrane1 Structure having lateral dimensions much greater than its thickness, through which mass transfer may occur under a variety of driving forces. Molecular Weight Cut-off (MWCO) See Nominal Molecular Weight Limit. Nanofiltration1 Pressure-driven membrane-based separation process in which particles and dissolved molecules smaller than about 2 nm are rejected. Nucleotide Cut-off (NCO) The number of nucleotides in a DNA fragment (single- or double-stranded) at which 90% of the fragment is retained by the membrane. Nominal Molecular Weight Limit (NMWL) The molecular weight at which at least 90% of a globular solute of that MW is retained by the membrane. Permeate (Filtrate, Ultrafiltrate) The solution passing through the membrane, containing solvent and solutes not retained by the membrane. Plugging Accumulation of debris on the fluid flow path, restricting or blocking flow. Rejection The fraction of solute held back by the membrane. Can be measured at any point in the process or averaged over the run. Retentate (Reject Stream, Concentrate) The solution containing the retained (rejected) species. Retention Factor1 (rF) Parameter defined as one minus the ratio of permeate concentration to the retentate concentration of a component. Note: rF = 1 [p]/[r] where [p] = concentration of solute in permeate, and [r] = concentration of solute in retentate. Reverse Osmosis1 Liquid-phase pressure-driven separation process in which applied transmembrane pressure causes selective movement of solvent against its osmotic pressure difference. Tangential Flow Filtration (TFF) Flow through a membrane device in which the fluid on the upstream side moves parallel to the membrane surface. Transmembrane Pressure (TMP) The driving force in ultrafiltration. In a stirred cell, equivalent to gas pressure. In centrifugal devices, it is related to g-force. In a flowing system, TMP decreases as the stream moves from inlet to outlet. Average TMP = [(Pin + Pout)/2] Ppermeate Ultrafiltration1 Pressure-driven membrane-based separation process in which particles and dissolved molecules smaller than 0.1 m and larger than about 2 nm are rejected. Yield Amount of species recovered at the end of the process as a percentage of the amount present in the feed solution.
References
1. Terminology for membranes and membrane processes (IUPAC Recommendations 1996). IUPAC, Journal of Membrane Science.1996;120(1): 14959.
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Acknowledgements
Millipore also wishes to acknowledge the following individuals for their contributions: Eduardo Vottero University of British Columbia Peter A. Lemaire and Dr. James Cole University of Connecticut Gary Smejkal Proteome Systems Woburn, MA Leonid Kryazhev Genome Quebec Montreal, Canada Mathew L. Thakur Thomas Jefferson University Hospital Philadelphia, PA Mark Merchant Helena Laboratories Beaumont, TX
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Department of Forensic Biology New York City Patrick O. Brown, Max Diehn, Ash Alizadeh Stanford University School of Medicine Dr. Sophia N. Karagiannis GKT School of Biomedical Sciences London
Patent
PCR is covered by US patents issued to Hoffmann-LaRoche, Inc .
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Lit. No. TP0040EN00 Rev. C 05/08 Printed in U.S.A. BS GEN-08-00329 2008 Millipore Corporation, Billerica, MA 01821 U.S.A. All rights reserved.