Blocking the Western Blot Membrane

The western blot technique relies on the fact that cellular proteins will bind to the nitrocellulose and PVDF membranes. Antibodies are also proteins, and they can also bind non-specifically to the membranes. To counteract this problem, membranes have to be “blocked” to prevent non-specific antibody interactions.

There are several classic proteins used to block western blot membranes. We will discuss all of the traditional blocking agents, along with their positive and negative features. We will also discuss the availability of non-protein blocking agents which are now commercially available from several biotech companies.

Blocking Protocol

  1. When the transfer is complete, remove the PVDF membrane and place directly in TBST (Be careful not to let the membrane dry at this point.) This step removes any excess methanol from the blot.
  2. Place the PVDF membrane into a fresh tray with your choice of blocking buffer. We recommend 5% BSA in TBST for most applications.
  3. Incubate the membrane in blocking buffer for at least two hours with gentle agitation on a shaker.
Bovine Serum Albumin

Bovine serum albumin (BSA) is commonly used as a blocking agent for all western blots. It is inexpensive and effective for blocking both nitrocellulose and PVDF membranes. Generally, BSA is dissolved to a 5% concentration in either TBST or PBST and incubated with a membrane for 2 to 16 hours. BSA is particularly useful for blocking membranes for phospho-protein western blotting. However, keep in mind that some Fraction V BSA has tyrosine phosphorylations and will give a high background with phospho-tyrosine motif antibodies.

To make 5% BSA in TBST (100 ml):

  • Add 5 grams for BSA to 100 ml of TBST.
  • Stir on a stir plate until BSA is completely dissolved.
  • Solution can be filtered to remove any particulates, although this is usually unnecessary if you are using quality BSA.

Non-Fat Milk

Non-fat milk is another extremely common blocking agent. Like BSA, it is inexpensive and effective for the vast majority of applications. Some commercially available blocking buffers actually isolate the casein protein from milk to use as a blocking agent. In contrast to BSA, however, milk and the milk protein casein are not ideal for blocking membranes for phospho-protein western blotting because casein is phosphorylated. Blocking with milk or casein, therefore, may produce a high background when blotting with anti-phospho-protein antibodies. Otherwise, milk and casein are best used at 1 to 2.5% W/V in TBST or PBST.

To make 5% Milk in TBST:

  • Add 5 grams of non-fat dry milk to TBST solution.
  • Stir until milk is completely dissolved and store at 4°C.

Fish gelatin can also be used to block western blot membranes. It is more expensive than both BSA and milk. Gelatin can decrease the background for many applications, but can also mask some antigens and decrease the sensitivity of detection. Generally, this is not the best option to use for standard western blotting techniques.


Normal serum can also be used as a blocking agent for western blotting, although it is more commonly used in immunohistochemistry methods. Serum contains the normal array of serum proteins that can saturate non-specific interactions during western blotting.

Protein-Free Blocking Buffers

A new product from most biotech companies is protein free blocking buffer. The idea behind these blocking agents is that protein-based methods always contain the possibility of cross-reaction with various antibodies. Protein-free blocking buffers, it is thought, can circumvent this problem by providing competition for non-specific sites while showing no cross-reactivity for blotting antibodies.

While this hypothesis seems to make sense, our experience is that BSA and milk work perfectly well for the vast majority of western blots. Further, because protein-free buffers can be up to 50% more expensive than traditional blocking agents, they should be considered only after other options have been exhausted.


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