Tubulin or Not Tubulin: Heading Toward Total Protein Staining as Loading Control in Western Blots

Western blotting is an analytical method widely used for detecting and (semi‐)quantifying specific proteins in given samples. Western blots are continuously applied and developed by the protein community. This review article focuses on a significant, but not yet well‐established, improvement concerning the internal loading control as a prerequisite to accurately quantifying Western blots. Currently, housekeeping proteins (HKPs) like actin, tubulin, or GAPDH are often used to check for equal loading or to compensate potential loading differences. However, this loading control has multiple drawbacks. Staining of the total protein on the blotting membrane has emerged as a better loading control. Total protein staining (TPS) represents the actual loading amount more accurately than HKPs due to minor technical and biological variation. Further, the broad dynamic range of TPS solves the issue of HKPs that commonly fail to show loading differences above small loading amounts of 0.5–10 μg. Although these and further significant advantages have been demonstrated over the past 10 years, only a small percentage of laboratories take advantage of it. The objective of this review article is to collect and compare information about TPS options and to invite users to reconsider their applied loading control. Nine benefits of TPS are discussed and seven different variants are critically evaluated by comparing technical details. Consequently, this review article offers an orientation in selecting the appropriate staining type. I conclude that TPS should be the preferred loading control in future Western blot approaches.