Antibody Purification Methods

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Antibody Purification Overview

Antibodies specific for an antigen of interest are one of the most useful and important tools that biology researchers can possess.The production and use of specific antibodies as detection probes and purification ligands (i.e., immunotechnology) has revolutionized bioresearch and diagnostic technologies.

Animals immunized with prepared antigens will produce specific antibodies against the antigen.When purified from serum or hybridoma cell lines that are prepared from tissue of the immunized animal, the antibody can be used directly (or after labeling with enzyme or fluorescent tags) to probe the specific antigen in Western blotting, ELISA or a variety of other applications. Antibodies are most commonly purified by one of two affinity purification methods.

Antibody Capture Methods

Because antibodies have predictable structure, including relatively invariant domains, it has been possible to identify certain protein ligands that are capable of binding generally to antibodies, regardless of the antibody's specificity to antigen. Protein A, Protein G and Protein L are three bacterial proteins whose antibody-binding properties have been well characterized.These proteins have been produced recombinantly and used routinely for affinity purification of key antibody types from a variety of species.These antibody-binding proteins are available immobilized to beaded agarose resin.

Proteins A, G and L have unique properties, which make each one suitable for different types of antibody targets (e.g., antibody subclass or animal species). It is important to realize that use of Protein A, G or L results in purification of general immunoglobulin from a crude sample. Depending on the sample source, antigens-pecific antibody may account for only a small portion of the total immunoglobulin in the sample. For example, generally only 2-5% of total IgG in mouse serum is specific for the antigen used to immunize the animal.

The interaction between the various proteins and IgG is not equivalent for all species or all antibody subclasses. The tables on the following page will help you decide which affinity protein is best for your application.


Figure 1. Binding sites of antibody-binding proteins.

Proteins used to immobilize antibodies to beaded support show specificity to different antibody domains. Protein A and G bind to the heavy chains of the antibody Fc region, while Protein L specifically binds the light chains of the two Fab regions of the F(ab')2 antibody fragment. *Protein G can also bind Fab fragments in certain conditions.


Table 1. Characteristics of immunoglobulin-binding proteins.
  Recombinant Protein A Recombinant Protein G Recombinant Protein L
Native Source Staphylo-coccus aureus


Peptostrepto-coccus magnus
Production Source E. coli E. coli E. coli
Molecular Weight 44,600 21,600 35,800
Apparent Mass by SDS-PAGE 45kDa 32kDa 36kDa
Binding Sites for Ig 5 2 4
Albumin Binding Site No No No
Optimal Binding pH 8.2 5 7.5
Ig Binding Target Fc Fc VL-kappa

The three proteins bind almost exclusively with the IgG class of antibodies, but their binding properties differ among species and subclasses of IgG. Protein A is generally preferred for rabbit, pig, dog and cat IgG. Protein G has better binding capacity for a broader range of mouse and human IgG subclasses (IgG1, IgG2, etc.).Protein L binds to certain immunoglobulin kappa light chains. Because kappa light chains occur in members of all classes of immunoglobulin (i.e., IgG, IgM, IgA, IgE and IgD), Protein L can purify these different classes of antibody. However, only those antibodies within each class that possess the appropriate kappa light chains will bind. Generally, empirical testing is required to determine if Protein L is effective for purifying a particular antibody.

Table 2. Binding characteristics of immunoglobulin-binding proteins

*Binding to Protein L will occur only if the immunoglobulin has the appropriate kappa light chains. The stated binding affinity refers only to species and subtypes with appropriate kappa light chains. Lambda light chains and some kappa light chains will not bind.

Antibody Polish Methods

As discussed above, a single, rapid capture step using affinity chromatography is often sufficient to achieve the level of purity and quantity of product required for research purposes. Antibodies or their fragments can be adequately purified for further use, and a polishing step is sufficient to remove unwanted impurity. If affinity chromatography cannot be used, or if a higher degree of purity is required, alternative techniques need to be combined effectively into a polish strategy. A significant advantage when working with native or recombinant antibodies or fragments is that there is often considerable information available about the product and contaminants, as shown in Table 3.

Table 3. Characteristics of IgG
Molecular weight Mr 150 000–160 000
Isoelectric point (pI) 4–9, most > 6.0, often more basic than other serum proteins
Hydrophobicity IgG is more hydrophobic than many other proteins
Solubility IgG very soluble in aqueous buffers. Lowest solubility (specific to each antibody) near pI or in very low salt concentration.
Temperature stability Relatively stable at room temperature (but specific to each antibody)
pH stability Relatively stable at room temperature (but specific to each antibody)
Carbohydrate Content 2–3%, most carbohydrate is associated with Fc region of the heavy chains

Antibodies are purified using purification techniques that separate according to differences in specific properties, as shown in Table 4.

Table 4. Antibody properties used during purification
Protein property Technique
Charge Ion exchange (IEX)
Size Gel filtration (GF)
Hydrophobicity Hydrophobic interaction (HIC)
Biorecognition (ligand specificity) Affinity (AC)

Every chromatographic technique offers a balance between resolution, capacity, speed and recovery. as shown in Figure 2.

Capacity, in the simple model shown, refers to the amount of target protein loaded during purification. In some cases the amount of sample that can be loaded will be limited by volume (as in gel filtration) or by large amounts of contaminants rather than the amount of the target protein.

Speed is most important at the beginning of purification where contaminants, such as proteases, must be removed as quickly as possible.

Recovery becomes increasingly important as the purification proceeds because of the increased value of the purified product. Recovery is influenced by destructive processes in the sample and by unfavorable conditions on the column.

Resolution is achieved by the selectivity of the technique and the efficiency of the chromatography matrix in producing narrow peaks. In general, resolution is most difficult to achieve in the final stages of purification when impurities and target protein are likely to have very similar properties.

Choose logical combinations of purification techniques based on the main benefits of the technique and the condition of the sample at the beginning or end of each step.

A guide to the suitability of each purification technique for polish is shown in Table 5.

Table 5. Suitability of purification techniques

Minimize sample handling between purification steps by combining techniques to avoid the need for sample conditioning. The product should be eluted from the first column in conditions suitable for the start conditions of the next column (see Table 5).


Antibody Purification

 Antibody Purification Tools

   Antibody Purification Agarose

  •  Protein A agarose beads (resin) for Antibody purification

  •  Protein G agarose beads (resin) for Antibody purification

  •  Protein L agarose beads (resin) for Antibody purification

    Antibody Purification Protein

  •  Protein A for Antibody purification

  •  Protein G for Antibody purification

  •  Protein L for Antibody purification

 Antibody Purification Technologies & Methods

   Antigen Affinity Purification

   Affinity Purification Principle

  •  Affinity Purification Principle by Protein A

  •  Affinity Purification Principle by Protein L

  •  Affinity Purification Principle by Protein G

   Antibody Concentration Methods

  •  Antibody Concentration Methods by Crystallization

  •  Antibody Concentration Methods by Precipitation

  •  Antibody Concentration Methods by Lyophilization

  •  Antibody Concentration Methods by Ultrafiltration

   Antibody Purification Methods

   Antibody Polish Methods

  •  Antibody Polish Methods by Hydrophobic Interaction Chromatography

  •  Antibody Polish Methods by Ion Exchange Chromatography

  •  Antibody Polish Methods by Size Exclusion Chromatography


 Related Links:

   Protein L/G/A Characteristics and IgG Binding Properties

  •  Protein L Characteristics and IgG Binding Properties

  •  Protein G Characteristics and IgG Binding Properties

  •  Protein A Characteristics and IgG Binding Properties


  •  Antibody Production Services

  •  Antibody Development Services

  •  Protein Production Services

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