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Western Blot Blocking Buffer Optimization

BSA vs nonfat dry milk

The blocking step of Western blotting prevents nonspecific binding of the antibody to the membrane by saturating the membrane with nonreactive proteins. The most common blocking buffers include 3-5% bovine serum albumin (BSA) or nonfat dried milk in a phosphate buffered saline (PBS) solution or tris buffered saline (TBS) solution. Small amounts of the detergent Tween 20 can be added to blocking solutions and washing solutions to further reduce background staining.

To obtain the best results from your western blot, you must decide which type of blocking buffer to use based on the detection system used and the antigen you’re trying to detect. For example, the phosphate in PBS interferes with alkaline phosphate detection systems, making TBS blocking buffer the better choice.

Buffer Advantages Disadvantages
BSA blocking buffer -Compatible with all detection systems and antibodies
-Allows for higher sensitivity detection
-Doesn’t inhibit nonspecific antibody binding
-Less complete blocking
-More expensive
Nonfat dry milk blocking buffer -Inexpensive
-More complete blocking
-Can reduce nonspecific antibody binding
-Incompatible with phospho-antibodies
-Incompatible with avadin/biotin detection systems
Boster TBS blocking buffer -Least expensive
-Compatible with all antibodies and detection systems
-Allows high sensitivity detection
-Ships as a powder
-Cannot be swapped into a different buffer (e.g. PBS for TBS)

Keywords: Western blotting optimization, blocking buffer, bsa, nonfat milk, pbs, tbs, best blocking buffer

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