The western blot, also sometimes referred to as the immunoblot, involves separating native or denatured proteins by gel electrophoresis, transferring these separated proteins to a protein binding membrane and subsequent detection of a target protein by an antibody specific to the target protein ( Figure 1). Although western blotting is a powerful and indispensable scientific technique that can be used to accurately quantify relative protein levels, it is necessary that proper experimental techniques and strategies are employed. These strategies include selecting the best normalization standard, proper sample preparation, determining the linear range for antibodies and protein stains relevant to the sample of interest, confirming the quality of the primary antibody, preventing signal saturation and accurately quantifying the signal intensity of the target protein.
In this manuscript we review essential strategies for improving confidence in the accuracy of western blots. This is not surprising since many labs are unaware of the limitations of western blotting.
Western blotting is one of the most commonly used laboratory techniques for identifying proteins and semi-quantifying protein amounts, however, several recent findings suggest that western blots may not be as reliable as previously assumed.
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