One of the key factors determining good western blot is in protein sample preparation. Where proteins can be obtained from nature, lab production, or extracted cells or tissues, proper preparation is compulsory. Extraction process starts from lysing cells or tissues using lysis buffer.

Many lysis buffer recipes are available for western blot purpose. But, choosing the one that suits your need require understanding of the protein you want to extract. Location of protein, structure of protein, protein solubility, and protein stability determine which buffer to use.

Lysis buffer with ionic detergent such as sodium dodecyl sulfate (SDS) can denature protein while others can maintain protein structure (native). Triton X-100 is considered weaker denaturant than SDS. Denaturing protein can lead to higher yield but some antibodies only work on native protein. However, some proteins are not soluble and need denaturing lyse buffer. Consider also the location of protein extracted, whether it is on cell surface, in membrane, in cytoplasm, or in whole cell. Proteins that are membrane bound need SDS in the buffer. Extracted proteins are susceptible to protease and phosphatase.

In order to eliminate protease and phosphatase effect, inhibitors are added to lyse buffer. Widely used inhibitors are phenylmethylsulfonyl fluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA) for protease. In addition, Na-fluoride and Na-orthovanadate are used against serine and tyrosine phosphatases respectively.

Antibodies are specific to their protein target in western blot. They cannot recognize proteins that are not in the appropriate form or degraded. Therefore, it is important to carefully choose the best lyse buffer during proteins extraction.