Immunopurification experiments

Summary

Antibody affinity purification is one of the most effective methods in protein purification. This step alone often results in 1000 to 10,000-fold purification. If the specificity of antibody binding to the target protein is demonstrated and the eluted target protein is not irreversibly damaged, then immunopurification can be considered an excellent protein purification method, especially when used in the final step of the purification process. Source: Handbook of Protein Technology

Operation method

immunopurification

Principle

Preparation of immunoaffinity columns requires that the antibody is first covalently bound to the medium. Alternatively, the antibody is bound to protein A or G beads, which are then solidified with a cross-linking agent. In the latter method, since the protein A or G binds to the Fe region of the body, the site of antigen binding is fully exposed and easily accessible. Once the immunoaffinity column is prepared, the antigen solution is ready for sampling, followed by washing away contaminants and then eluting the antigen. If it is not possible to use specific ligands to destroy the interaction between the antigen and antibody, a non-specific elution method is used. However, non-specific elution methods must be applied carefully to avoid inactivation of the target protein. Monoclonal antibodies are often more useful in immunoaffinity purification than polyclonal antibodies. A monoclonal antibody represents a homogeneous antibody with a single specific binding site to the target protein, and thus its binding is deterministically consistent. The same is true for elution, since only a single type of interaction needs to be disrupted to release the target protein. It should be noted that polyclonal antibodies are also usable in affinity purification, especially those produced by immunizing animals with very pure antigens. In summary, the main key to performing an effective immunopurification is to obtain a specific antibody, which has YI a certain strength of affinity binding to the substrate. This strength of binding force on the one hand can keep the substrate protein still bound to the column during the process of washing, not to be eluted; on the other hand, the binding is not too tight and difficult to elute, using extreme elution conditions caused by the denaturation of the substrate protein. Therefore, the method of selecting antibodies for immunopurification should be to test a set of monoclonal antibodies and select the ones that not only have sufficient affinity binding, but also can elute the complete antigen. The quality of the immunopurification depends on the purity of the antibody solution. Antibodies used in the preparation of immunoaffinity columns should have both the previously mentioned properties and be as pure as possible. Immunoaffinity columns are quite expensive to prepare and have a relatively low binding capacity, often binding up to 1 mg of antigen per ml of media. Therefore, in order to save and increase efficiency, smaller and thicker columns are often prepared, while the sample solution is required to be repeated several times during the affinity purification step in order to purify more antigenic proteins. In addition, smaller columns can also reduce and limit antibody loss due to column contamination or inactivation by egg-water enzymes. Immunoprecipitation can also be viewed as another type of immunization and purification. Still another type is reverse immunopurification, which uses an antibody column to remove a specific contaminant an antigen from solution.

Materials and Instruments

Antigen
PBS HCl NaN3
Centrifuge

Move

1. 5 to 10 times the volume of the starting buffer, e.g., phosphate buffered saline (PBS);

2. 3 to 5 times the column volume of elution buffer, such as 0.1 mol/L, pH 2.5 glycine-HCl to wash away contaminants;

3. equilibrate the column with 5-10 times the column volume of the starting buffer;

4. Equilibrate the sample with the starter buffer by dialysis, gel filtration, and addition of 10× starter buffer (1/10 volume) to the sample solution. The sample should be centrifuged at 100,000 × g for 30 min or filtered through a 0.22 um membrane;

5. Apply the sample to the column. A slower flow rate will result in better antigen binding. The sample should be cycled several times;

6. Wash the column with 5-10 times the volume of Aira buffer or until the _A280 value reaches the baseline or background level. If the antigen-antibody interaction is strong, the starting buffer can contain a certain concentration of salt, such as 0.5 mol/L KCl, to reduce non-specific binding;

7. 2-fold volume of elution buffer, e.g., 0.1 mol/L, pH 2.5 glycine-HCl, washes the column and collects the eluate in 1-ml fractions. The low pH of the eluent is immediately neutralized by adding 0.1 ml of 1 mol/L Tris-HCl, pH 8.0, to each collection tube. 8;

8. test the fractions and combine the active fractions;

9. dialysis or gel filtration if the limited stability of the antigen requires immediate change of buffer;

10. 10x volume of 0.2 mol/L, pH 2.5 glycine-HCl, wash the column, followed by 10-20x volume of PBS, or 0.02% _NaN3 in PBS for storage.

Caveat

The ligand should be stable in the pH conditions of the buffer used. Do not allow the solution in the column to run dry. All solutions should be filtered and decontaminated through a 0.22 um membrane to maximize column life. The antigen binding capacity of the media can be determined in a test tube. The test is performed by adding a constant amount of antibody media and incremental amounts of antigen to a set of test tubes, mixing them, and then determining the binding capacity.

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