Since its first introduction by Towbin, et al in 1979, western blotting (WB) is widely applied to detect the existence of specific proteins from a mixture, evaluate the size of the protein of interest, and measure the level protein expression. The process of WB includes denaturing the protein mixtures and separating them based on molecular weight via gel electrophoresis, transferring the separated bands to a blotting membrane, after blocking the remaining surface of the membrane, recognizing the protein of interest with a primary antibody and detecting by the secondary antibody.
WB can be a quick and dirty way to test cell lysate during the protein expression to see if the protein of interest is expressed and what is the expression level looks like. It would be important to check pellet from P2 expansion for baculovirus production or E coli expression pellet before purification. In addition, impurities information can be easily obtained from the WB, such as level of protein contaminant, if the contaminant band by SDS-PAGE is non-specific carry-over or degradation of products. Furthermore, when antibody is the protein of interest for expression, WB can demonstrate the functional expression of heavy and light chains with the appropriate molecular weight, and they are paired correctly.
WB is considered as an essential QC for many cases. For example, proteins used in in vitro assays, where detection is by antibody to tag or the protein itself, the WB validation is critical. For ELISA data submitted to reviewers of regulators and peer-reviewed journals, WB is also widely accepted as a minimal confirmation QC step to confirm the protein used is the correct one.
The limitations of WB due its poor reproducibility and error prone for quantification purpose. A primary antibody detects specifically to the protein of interest is the key. Applying WB is sometimes limited by the lack of fully validated antibody that is against the protein not the tags. Depending on the expression systems used (E. coli, bacillus, HEK293, CHO, and others), in some cases, there can be non-specific binding of other proteins to the tags used in expression, so thorough characterization to the express system “background noise” is desired. Proteins with high molecular weight are poorly resolved on gels make it more difficult to perform WB. Thus, additional protein QC and characterization methods are desired to obtain more information on protein purify and identification.
Please stay tuned for our next blog in the series to find out more on the additional protein QC and characterizations that your protein expression needs.
