Blocking buffers have an essential role to play and they are often considered as the third wheel in the relationship of antigen and/or antibody. By opting for the right blocking buffer, you can enhance the ability of the antibody to unite/bind its specific antigen while the bad blocking can help keeping the definite antibody as far as possible. If you don’t want bad blocking to distort your blotting experiments, then it’s imperative to understand what your blocking buffer can help you attain and how you can come up with the best blocking buffer.

The ultimate goal of the blocking buffer is to cover or shield the membrane surface that doesn’t have a protein attached to it. If proper blocking is not allowed to take place, the spaces will seize the antibodies and cause non-specific binding. Non-specific binding can cause potentially produce false positive signals to the experiment, also known as ‘noise’ or ‘background’.

Here are some of the most commonly used blocking buffers and some guidelines as to how you can use them:

Skim Milk Powder/Non-Fat Dry Milk Powder

Skim milk powder is by far the cheapest and most commonly available blocking buffer on this list. You can easily buy it from a supermarket or from a laboratory nearby. Generally, skim milk powder is used as a blocking buffer in solutions ranging 2-5%. However, one important consideration is that milk naturally contains phosphoproteins; hence, it mustn’t be used if you are utilizing phospho-specific antibodies.

Bovine Serum Albumin (BVA)

This blocking buffer is basically the purified version of Albumin which consists of a portion of bovine serum, as well as skim milk powder. This combination is one of the commonly utilized blocking buffers. In most cases, they have a concentration of 2.5%, but researchers fluctuate with it on account of the protocol they use as they are expensive. However, make sure you don’t use BVA in blotting experiments with IgG or serum proteins if you don’t want the noise to thrust aside your efforts.

Fish Gelatin

Fish gelatin is the evolved version of what we knew as porcine gelatin, a commonly used blocking buffer. Fish gelatin is derived and purified from the membrane of the cold-water fish. They have a unique trait and ability to maintain their liquid state even under cold temperatures. Typically, they are utilized at reduced concentrations like 0.1-5%. Unlike skim milk powder and BVA, fish gelatin cannot cross-react with mammalian antibodies as they do not consist of serum proteins. As a result, there is no chance of ‘noise’ or ‘background’ to take place.

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