The use of membrane blocking agents in Western Blot is a critical step to prevent non-specific binding of our antibody, blocking the points of the membrane that do not contain ligand.
The most frequently used Western Blot membrane blocking agents are BSA (Albumin from bovine serum) or milk. But what differences, advantages and disadvantages are there between using one or the other?
The choice between bovine serum albumin (BSA) and skimmed milk depends on generic factors such as price or availability, but also on more specific criteria such as the type of protein we are analyzing, its level of expression or the binding strength of the antibody. We analyze it below.
BSA OR MILK TO BLOCK THE MEMBRANE IN WESTERN BLOT?
As we mentioned in the introduction, the first criterion to take into account when selecting the blocking agent that best fits our Westren Blot experiment is the binding capacity of the antibody and the level of expression of the protein.
Broadly speaking, milk is the blocking agent of choice when we want to adjust the budget and resort to a reagent of greater availability, as long as the antibodies we are using have a good capacity for binding to the antigen, and the protein of interest is find at relatively high levels in the sample.
In contrast, BSA (bovine serum albumin) is usually used in the case of antibodies with a more limited binding capacity or when we have a low amount of protein in the test sample.
In the second place , we must bear in mind that while the BSA is a single purified protein, milk is composed of a mixture of different proteins. Therefore, in the case of using phosphospecific antibodies , it is recommended to use BSA as a membrane blocking agent in western blotting, because milk contains phosphorylated proteins (eg casein) that could react nonspecifically with the antibody.
ADVANTAGES AND DISADVANTAGES OF USING BSA AGAINST MILK
That said, we can summarize the advantages and disadvantages of using BSA vs Milk as membrane blocking agents in Western Blot as follows:
|BSA||· Lower risk of cross reactivity|
· More specific and cleaner results
· Allows its use with phosphorylated proteins
· Suitable for use with proteins present at low levels
|· Higher cost product|
· The carbohydrates it contains can increase the background noise in the presence of lectin
|MILK||· More economical|
· Greater blocking capacity
· Easy to acquire and prepare
|· Not recommended for use with phosphospecific antibodies|
Cannot be used in streptavidin-biotin-based experiments (milk contains biotin)
· Reduces the sensitivity of some commercial anti-HIS antibodies
· Can mask proteins found in low amounts in the sample