Commonly used blocking solution for Western blot and precautions

In the Western blot experiment, a solid phase carrier (such as NC membrane, PVDF membrane) is used. On the surface of these solid phase carriers, there are many holes. By electroporation, the protein on the gel is transferred to the membrane, and the protein is mechanically filled. (Stacking) and adsorption are combined with the surface. The protein is stuffed into many holes in the surface, but the protein is not continuous, but there are many gaps, the antibody is also a protein, and it will be adsorbed in the empty hole, so there will be many non-specific signals.

The protein in the blocking solution can be combined with the blank position on the surface of the solid phase carrier, and also bonded to the membrane by mechanical filling (stacking) and adsorption coating, because there is a "filling" and covering the "protein binding site to avoid the primary antibody. Non-specific binding, so there is a "closed" statement. These two effects can make the protein in the blocking solution firmly bind to the blank position, so that the antibody protein will not be non-specifically adsorbed onto the membrane, but only Will bind to specific proteins.

The blocking solution should block all unbound sites without replacing the target protein on the surface, not binding to the target protein epitope, or cross-reacting with the antibody or detection reagent.

Common blocking solution:

BSA is the most commonly used blocking solution and the ingredients are suitable for most situations. If the immunogen is BSA-conjugated, BSA has strong immunogenicity, which will lead to the production of many antibodies against BSA. In order to avoid cross-reaction, BSA can not be blocked with skim milk powder. Casein or protein-free blocking solution can be used. .

There are two main principles for blocking serum. The first is that some of the samples to be tested are non-specifically bound to the protein, which leads to too high background. Therefore, BSA or serum can be used to block this part-specific binding. From this point of view There was no significant difference between BSA and serum. The second sample to be tested may have an Fc receptor, which can bind to the constant region of the antibody, and has no antibody specificity. The antibody is blocked in the serum, so that the primary antibody and the secondary antibody and the sample Fc receptor can be blocked by the serum. The combination of BSA does not block the Fc receptor.

The biggest advantage of skimmed milk powder is that it is cheap, but because of its relatively complex composition, the scope of application is narrower. Phosphorylated antibodies may cause an increase in background and, in addition, because they contain biotin, they cannot be used in biotinylated antibody systems. Skim milk powders contain small amounts of biotin and alkaline phosphatase residues, which can also increase high background or background levels when using the biotin-avidin system and alkaline phosphatase-labeled secondary antibodies.