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Affinity interaction chromatography is often the single most effective step in any protein purification procedure. Up to 95% purity can be achieved in one step, depending on the nature of the interaction and the starting composition of the protein solution. Well known examples of highly specific affinity interactions include antibodies and Protein A/G; multiple histidine tags and nickel; streptavidin and biotin; antibodies and antigens; and many others. Less specific interactions are also used for enrichment or depletion protocols, including albumin depletion on cibacron blue resin, glycoprotein enrichment on concavalin A resin, and capture of nucleic acid-binding proteins on heparin resin.
Antibody purification is performed to concentrate and enrich antigen-specific antibodies and lower background by removing any non-specific proteins. This step is performed when highly purified antibodies are required, such as in immunohistochemistry and immunocytochemistry. Affinity purification of monoclonal and polyclonal antibodies most commonly uses Protein A and Protein G chromatography. The Montage Antibody Purification Kit with PROSEP-A/G media is designed for simple and rapid antibody purification by centrifugation from serum, ascites, or cell culture supernatants.
The Montage Antibody Purification Kit incorporates pre-packed Protein A or Protein G media plugs and spin columns, along with the buffers and filtration devices necessary for pre-clarification of the source material and concentration of the purified antibody. Spin-columns with PROSEP-A or PROSEP-G media are also available separately.
Antibody samples purified using Montage spin columns may be used in a wide range of laboratory procedures such as immunoprecipitation, immunofluorescence, Western blotting, ELISA, and others. The antibodies can be used for radiolabelling, conjugations (e.g., fluorescein), or preparation of immuno-affinity columns.
The Montage Antibody Purification Kit with PROSEP Media contains:
This section provides information that may be helpful when preparing to purify antibodies. Sections will include:
Immunoglobulin G from most species consists of several subclasses with different biological properties. Four subclasses of IgG have been identified in human (IgG1, IgG2, IgG3, and IgG4) and in mouse (IgG1, IgG2a, IgG2b and IgG3). For immunological studies, it is often necessary to isolate one particular subclass of IgG from the other subclasses.
Protein G binds to all major Ig classes except IgM and therefore has a wider reactivity profile than Protein A. However, the binding of IgGs to Protein G is often stronger, making elution and complete recovery of the immunoglobulin more difficult. Interestingly, due to the lower cost of Protein A compared to Protein G, researchers tend to experiment first with Protein A, then Protein G. Protein A withstands harsher conditions used in cleaning and regeneration.
The affinity of interaction of Protein A with mouse IgG subclasses varies. The most common subclass of mouse monoclonal antibodies is IgG1. Customization of the purification strategy may be required for the affinity separation as mouse IgG1 does not generally bind well to Protein A. However, as the affinity interaction is pH and salt dependent, under high salt regimes (2-3 M NaCl) and high pH (pH 8-9), the antibodies will bind to Protein A.
|Immuoglobin Heavy Chain||Mean serum concentration (mg/mL)||Sedimen-tation constant||Molecular weight||Molecular weight of heavy chain||Number of heavy chain domains||% Carbohydrate|
|slgA a1 or a2||0.05||11S||385,000||52-56,000||4||7-11|
|Human IgG1||++||++||Rat lgM||+/-||-|
|Human IgG2||++||++||Rabbit lgG||++||++|
|Human IgG3||-||++||Hamster lgG||+||++|
|Human IgG4||++||++||Guinea Pig lgG||++||+|
|Human IgA||+||-||Bovine lgG||+||+|
|Human IgD||+||-||Sheep lgG||+/-||+|
|Human IgE||+||-||Goat lgG||+/-||+|
|Human IgM||+||-||Pig lgG||++||++|
|Mouse IgG1||+||+||Chicken lgG||-||+/-|
|Mouse IgG2b||++||++||Human Fab||+||+|
|Mouse IgG3||+||++||Human F(ab’)2||+||+/-|
|Mouse IgM||+/-||-||Human ScFv||+||-|
|Rat IgG||++||++||Human Fc||+||-|
|Rat IgG1||+/-||+||Human K||-||-|
Protein A is a cell wall protein from Staphylococcus aureus with a molecular weight between 35-50 kDa. The quality of the Protein A media (or equivalent) is important to avoid leakage of Protein A during the elution procedure. PROSEP-A High Capacity media is based on a porous glass matrix that is fully incompressible yet highly porous, with a very high percentage of large, open-ended, interconnected pores. Its open pore structure allows very rapid mass transport, resulting in very high dynamic capacity for IgG. Immobilized Protein A binds specifically to the Fc region of immunoglobulin molecules of many mammalian species.
Protein A affinity chromatography is a rapid one-step purification, which removes most non-IgG contaminants and can achieve purities close to homogeneity. It is particularly useful for purifications of tissue culture supernatant, where 10- to 100-fold concentrations can be achieved.
Protein G is a cell wall protein from Streptococci with a molecular weight of 22 kDa. The covalent immobilization of the Protein G is important to avoid leakage of Protein G during the elution procedure. PROSEP-G consists of recombinant Protein G covalently immobilized on porous glass that is more rigid and durable than conventional polymeric matrices. Porous glass also has a very high uniform internal pore size distribution that falls within a very narrow range, resulting in uniform rates of diffusion and hence rapid mass transfer between solution and solid phase. The Protein G that is immobilized is a recombinant protein from which the albumin binding region has been deleted. Immobilized Protein G binds specifically to the Fc region of immunoglobulin molecules of many mammalian species. In many instances antibodies bind more strongly to Protein G than Protein A. PROSEP-G can be used to purify antibodies that may be difficult or impossible to purify on PROSEP-A High Capacity Media (for example, Rat IgG2b and Human IgG3). Excellent yields have been established using PROSEP-G resin with polyclonal rat, rabbit, and human antibodies.
Protein G affinity chromatography is a rapid one-step purification, which removes most non-IgG contaminants and can achieve purities close to homogeneity. It is particularly useful for purifications of tissue culture supernatant, where 10- to 100-fold concentrations can be achieved.
Any antibody-containing sample, such as a crude biological extract, a cell culture supernatant, serum, or ascites can be used in the Montage spin column. It is important that the sample is first filtered through a 0.22 :m Steriflip-GP filter unit to remove particulates that could clog the media flow channels. This is critical for re-use of the device. All samples should be filtered just prior to loading even if they have been filtered several days before the chromatographic run. Aggregation precipitation of proteins is common during storage and repeated freeze/thaw cycles in sera, ascites and tissue culture supernatants. Lipids, which can be found at high levels in serum or ascites, should also be removed (see Delipidation Procedure). Millipore recommends that samples from only one source be run on any given column. In PROSEP separations, the sample should simply be diluted 1:1 (v/v) in 1 x binding buffer.
The most common elution conditions for Protein G affinity separations involve a reduction in pH to pH 2.5. It is important to appreciate that a few proteins (e.g., some monoclonal antibodies) are acid-labile and they can lose their activity at very low pH values. Above all, the elution conditions must preserve the integrity and activity of the target protein. Most observed denaturation is caused by harsh elution conditions. Acidic pH is known to reduce the antibody titre, decrease immunoreactivity and distort the antibody structure. It is therefore critical that the pH is restored to neutrality immediately after elution.
Controlling the flow rate through an affinity chromatography support is important in achieving binding. Flow rate through the column support is inextricably related to the efficiency of the separation; too fast a flow will cause the mobile phase to move past the beads faster than the diffusion time necessary to reach the internal bead volume. Our studies demonstrate that the large internal surface area of the Montage media bed compensates completely for the velocity of the mobile phase through the column support when the centrifugal speed does not exceed 1,000 Hg* for PROSEP-G or 1,500 xg* for PROSEP-A. The PROSEP media chemistries used in the Montage spin column have sufficiently rapid association kinetics between the protein molecule and the immobilized ligand to allow for optimal diffusional flow through the internal bead structure. Traditionally, gravity flow chromatography is very slow and resolution of the protein separation can be adversely affected by secondary diffusion effects.
This section outlines the information necessary to purify antibodies using the Montage Antibody Purification Spin Columns with PROSEP Media, including information for those not using the kit about preparing the buffers. Other information will include:
Use the following recipes to prepare the buffers supplied with the Montage Antibody Purification Kit with PROSEP-G Media. All buffers contain 0.1 % sodium azide as a preservative and can be stored at room temperature.
After loading the plug into the spin column and placing the spin column into a centrifuge tube, follow the procedure below.
All Protein A and G affinity columns are affected by the presence of lipids and lipoproteins, especially in antibody samples derived from ascites fluid. For end users who have antibody solutions that they need to delipidate, the following protocol is a gentle and easy method for removing lipids and lipoproteins.
By using the Beer-Lambert law, A = e.c.l, the concentration of IgG (mg/mL) in the sample can be measured by multiplying the absorbance at 280 nm by 0.72. If IgM or IgA are purified, multiply the absorbance at 280 nm by 0.84 or 0.94, respectively. These antibody concentrations are only estimates as other contaminating proteins can also contribute to the absorbance reading. However, they can provide a reliable and quantitative method for determining the concentrations of pure antibody solutions. Most researchers use different sandwich ELISA assays to accurately measure antibody concentrations within ranges of 0.01 mg/mL to 20 mg/mL.
The antibodies are monitored for purity by SDS-PAGE under reducing or non-reducing conditions. Note that IgG appears in a reducing SDS-PAGE as 25 kDa and 50-55 kDa bands. Recovery of immunoglobulins can be quantified by a standard protein assay, scanning densitometry of reducing or non-reducing SDS-polyacrylamide gels or ELISA. Antigen binding parameters can be measured for both affinity and avidity.
Source: Recombinant Protein A or G expressed in E. coli Supporting matrix: Porous glass Media particle size: 74 :m-125 :m Media bed volume: 1. 6 mL Recommended working pH: pH 2.0-9.0 Binding capacity: for PROSEP-A typically >15 mg rabbit IgG and for PROSEP-G typically >8 mg rabbit IgG Solubility in water: Insoluble Maximum volume in swinging bucket rotor: 20 mL
All resins are susceptible to oxidative agents. Avoid high temperatures. Protein G is resistant to limited exposure to 8 M urea pH 10.5 and extremes of pH (e.g. pH 1.0 and pH 11). It lacks the robustness of Protein A with respect to 1 M NaOH and it is denatured under these aggressive conditions. However, Protein G is stable to treatment with 0.1 M NaOH.
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 μm. 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.