Abstract:    Blocking plays an important role in Western Blot. Thus, antibodies can more specifically bind with antigen by choosing suitable blocking buffer, which lays the foundation for successful WB as well. Although the operation of this step is simple, it's difficult for many scientific researchers to choose blocking buffer. Advantages and disadvantages of six blocking buffers are introduced below.
Keywords:  Western Blot Blocking Buffer, Western Blocking Buffer, Blocking Reagent, Western Blot Antibody

1. Skimmed Milk Powder(SMP)

Skimmed milk powder is the cheapest and easiest way to buy blocking buffer. It can be immediately used upon buying in chemical products company or supermarkets. The general concentration of blocking buffer is between 2.5-5%(w/v). Significantly, Skimmed milk powder is the mixture of many kinds of proteins, including casein which belongs to phosphoprotein. If your target protein is phosphoprotein, SMP shouldn't be selected. Otherwise, there are many background signals. Even though the target protein isn't phosphoprotein, it's easy to generate high background signals due to the protein mixture.

2. Bovine Serum Albumin(BSA)

Bovine serum albumin is a kind of globulin purified from bovine serum. Besides skimmed milk powder, it is a commonly used blocking buffer. The concentration of BSA blocking buffer is between 2-5%(w/v). The concentration can be changed according to your experimental requirement. Due to the high price, many researchers use the valid concentration as low as possible. BSA isn't phosphoprotein and is the single protein. Contaminations like IgG or other serum albumin may be contained during purification. These proteins can generate cross reaction with mammalian antibodiess and will increase the non-specific background signals.

3. Whole Serum

Whole serum is the mixture consisting of many proteins and can be also used as blocking buffer, e.g. fetal bovine serum(FBS), rabbit serum, goat serum etc. The usage is very low. Compared with skimmed milk powder and BSA, the advantage of whole serum isn't obvious. However, the price is higher. The general concentration is between 5-10%(w/v). Like BSA, the whole serum contains immunoglobulin and serum albumin. The cross reaction with mammalian antibodies will happen as well.

4. Isinglass

Developed from pigskin glue, isinglass is used as blocking buffer. Isinglass is extracted from the skin of pisces and won't be frozen in the low temperature. The general concentration is between 0.1-5%(w/v). Different from skimmed milk powder and BSA, isinglass doesn't contain any serum albumin. The cross reaction with mammalian antibodies won't occur. Background signals can be greatly decreased. However, isinglass can't be applied in blocking biotin-avidin antibody protein system, because it contains endogenous biotin.

5. Polyvinyl Pyrrolidone(PVP)

PVP is the non-protein surrenal of traditional blocking buffer. Although relevant articles were published in 1993, PVP is a relative new product in blocking technology. PVP is a kind of water soluble polymer, which can rapidly bind with nitrocellulose and PVDF membrane. The general concentration is between 0.5-2%(w/v). Usually, PVP is used with other blocking buffers. It is very efficient in detecting small target protein.

6. Commercial Blocking Buffer

Commercial blocking buffers usually contain purified protein. The advantage is that unavoidable ingredients in other blocking buffers like phosphoprotein, immunoglobulin, albumin, biotin etc aren't contained. Thus, the price is higher. Each commercial blocking buffer has its own protocol. The blocking time is very short by simply following the protocol.

7. Conclusion

Now, you have a comprehensive understanding in various blocking buffers. Above all, selection of blocking buffers is based on three things: antibody, target protein and detecting system. It's not easy to find the blocking buffer fitting the specific target protein. Besides excluding some obvious unsuitable products, it will take a lot of time to try various kinds of blocking buffers with different concentrations. FineTest offers western blot antibodies as well.