Western Blot Optimization:

BLOCKING BUFFER OPTIMIZATION

Choosing the right blocking buffer is key to a clean successful Western blot. If you run into background or specificity issues and suspect it is the blocking that failed you, use this guide to decide what better options you may have.

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BSA vs Non-Fat 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 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.

Each Blot May Require Different Blocking Buffer

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.

Advantages and Disadvantages of Western Blot Buffers

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