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In the Western blot experiments, solid-phase carriers (such as NC membranes, PVDF membranes) were used. There were many holes in the surface of these solid-phase carriers, and by electron transfer, the proteins on the gel were transferred to the membrane and the proteins were filled (stacked) and adsorbed to the surface. The protein into the surface of a lot of holes inside, but the protein is not continuous, and there are many gaps. Antibody is also a type of protein, will be adsorbed in the empty hole, so there will be a lot of non-specific signal.
The proteins in the blocking fluid can bind to the blank site of the surface of the solid support, and are also bound to the membrane in a manner that is mechanically filled (packed) and adsorbed, since there are “fill “and “cover”; protein binding sites to avoid primary non-specific binding, so there are called blocking solution. The same two effects of the protein in the blocking solution can be firmly combined in the blank position, so that the antibody protein will not be non-specific adsorption to the membrane, but only will be combined with specific protein.
The blocking fluid should block all unbound sites without replacing the target protein on the surface, not binding to the target protein epitope, nor cross-reacting with the antibody or detection reagent. This article describes some of the characteristics of the common closure of the liquid, help you make a decision!
BSA is the most commonly used blocking solution, the composition is simple and could be applied to most cases. If the immunogen is coupled with BSA, since BSA has a strong immunogenicity, it will lead to a lot of antibodies against BSA. In this case, to avoid cross-reaction, we should use skimmed milk powder as blocking. You can use casein or protein-free blocking fluid.
The principle of blocking serum is mainly two points. The first is that some of the protein and will non-specific bind to the samples, resulting in the background is too high. So, you can use BSA or serum to block this non-specific combination. From this point of view, BSA and serum were not significantly different. The second is that test sample may have Fc receptors, which can bind to the antibody constant region, independent of antibody specificity. There are antibodies in the serum. So, the serum can block the binding between primary antibody, secondary antibody and the sample Fc receptor while BSA cannot block the Fc receptor.
- Skimmed Milk Powder
The biggest advantage of skimmed milk powder is cheap, but the composition is relatively complex. So, the application of skimmed milk powder is narrower. Phosphorylated antibodies may lead to increased background, in addition, because they contain biotin, cannot be used for biotin-labeled antibody system. Skimmed milk powder contains a small amount of biotin and alkaline phosphatase residues, in the use of biotin-avidin system and alkaline phosphatase labeled secondary antibody, will also lead to high background or background level increased.