Western blotting and other immunological protein-analytical methods are widely used for detecting and (semi-)quantifying specific proteins in given samples. This webinar will discuss a significant, but not yet well-established, improvement concerning the internal loading control as a prerequisite to accurately quantifying proteins by these methods. Currently, housekeeping proteins (HKPs) like actin, tubulin, or GAPDH are often used to check for equal loading or to compensate potential loading differences. However, these loading controls have 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 15 years, only a small percentage of laboratories take advantage of it. The objective of this webinar is to collect and compare information about TPS options and to invite users to reconsider their applied loading control. While the focus of this webinar will be on Western blots, similarities with other immunological protein-analytical methods will be illustrated and Simple Western total protein assay will be discussed.

 

Learning Objectives