A Novel Method for Normalization in Western Blotting that is Superior to the Use of Housekeeping Proteins

Western blotting workflows have long relied upon the use of housekeeping proteins (HKPs) as a control for sample normalization in order to account for differences between sample loads. Unfortunately, HKPs often show signal saturation, can vary in response to the treatments being studied, or show variation in expression between different cell types leading to erroneous results. In contrast, new methods of normalization that involve Total Protein Normalization (TPN) have been gaining support. In this study we investigate a new method for TPN that utilizes the Invitrogen No-Stain Protein Labeling Reagent, which allows for all of the proteins on a membrane to be labeled uniformly and quickly while not interfering with downstream immunodetection methods. We compare this new method against common housekeeping proteins in four different cell backgrounds (HeLa, MCF-7, Jurkat, and A431 cells). Our data illustrate how common housekeeping proteins exhibit signal saturation and yield incorrect normalization data. The No-Stain TPN method shows superior protein normalization compared to the use of housekeeping proteins with decreased sample-to-sample variation (8% versus 48%). The No-Stain TPN method provides an elegant alternative for achieving accurate quantitative western blots. Support or Funding Information This work was supported by Thermo Fisher Scientific