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Greetings Earthling,
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 the meme for blocking optimization tips!)
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Antibody compatibility
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Compatibility of the protein of interest
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The detection system
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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:
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Nonfat dry milk is the most economic choice, but should be avoided for blots using biotin-conjugated antibodies due to intrinsic amounts of glycoprotein and biotin present in milk. Additionally, naturally present phosphatases may lead to protein dephosphorylation, interfering with target identification of phosphorylated proteins.
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BSA or Casein in TBS are recommended for phosphorylated target analysis or when using alkaline phosphatase-based detection methods. TBS buffer instead of PBS buffer should be chosen because PBS interferes alkaline phosphatase.
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Use of NaN3 as a preservative in blocking reagents should be avoided when using peroxidase detection systems due to its oxidase inhibiting properties.
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BSA will usually yield clearer results because it contains fewer cross-reactive proteins. However, some antibodies will work better with milk which contains a greater variety of blocking proteins.
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On the western blot, black spots can be the result of the antibody binding to the blocking agent. In this case, we suggest filtering the blocking agent to avoid this problem.
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We advise reviewing the data sheet for any recommended blocking agents because antibodies can be very sensitive to the blocking agents used.
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:
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