Western Blot Tips and Tricks: Blocking Optimization

Factors to Consider When Choosing the Right Blocking Agent for WB

Following protein transfer, it is important to block the unreacted sites on the membrane using inert proteins and/or nonionic detergent to reduce levels of nonspecific protein binding during the assay. Blocking buffers should block all unreacted sites without disrupting target protein-membrane interactions or affect epitope availability.

There are three factors to consider when choosing an appropriate blocking agent for your specific protocol (click here for more western blot blocking optimization tips):

Antibody compatibility
Compatibility of the protein of interest
The detection system

Advantages and Disadvantages of Blocking Agent

The most typical blocking solutions are nonfat dry milk, casein, gelatin, or Tween-20 in TBS and/or PBS buffers. Here are some advantages and disadvantages of some common blocking agents:

Blocking Agent Advantages:

Nonfat dry milk is the most economical choice for blocking.
BSA or Casein in TBS are recommended for phosphorylated target analysis or when using alkaline phosphatase-based detection methods.
BSA typically yields clearer results due to containing fewer cross-reactive proteins.
Filtering the blocking agent can prevent black spots on the western blot caused by antibody binding.

Blocking Agent Disadvantages:

Nonfat dry milk should be avoided for blots using biotin-conjugated antibodies due to intrinsic amounts of glycoprotein and biotin present in milk.
Naturally present phosphatases in nonfat dry milk may lead to protein dephosphorylation, interfering with target identification of phosphorylated proteins.
TBS buffer should be chosen over PBS buffer when using BSA or Casein due to PBS interfering with alkaline phosphatase.
The use of NaN3 as a preservative in blocking reagents should be avoided when using peroxidase detection systems due to its oxidase inhibiting properties.
Some antibodies may work better with milk due to its greater variety of blocking proteins.

We advise reviewing the data sheet for any recommended blocking agents because antibodies can be very sensitive to the blocking agents used.

A Step-by-Step Western Blot Experimental Protocol Workflow

Here is a summary workflow for a typical WB experimental protocol along with necessary reagents for each step. Click on the flowchart image to shop WB Reagents & Kits, 20.17% off for a limited time!

Many different factors in the blocking step may need troubleshooting during western blotting... For more troubleshooting and optimization tips, visit our technical resource center:

More WB Resources

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