Method for quantitative western blotting that eliminates errors from the use of housekeeping proteins
The western blotting technique that began as a means of detecting the protein of interest in a complex sample that may contain numerous other proteins has been in practice for four decades. In recent years, journal editors and reviewers have requested adoption of methods that improve the accuracy and reproducibility of quantitative western blot data. Protein normalization is a critical step in obtaining reliable and reproducible quantitative western blot data. Total protein normalization is considered the gold standard for quantitative western blotting. Protein normalization of western blots has relied upon housekeeping proteins, which exhibit signal saturation and varying levels of cellular expression. These issues can produce spurious results, leading to erroneous conclusions. In this webinar, we will talk about a new protein labeling reagent that offers an attractive alternative to achieve total protein normalization. The Invitrogen No-Stain Protein Labeling Reagent is a flexible, accurate, rapid, and reliable method to visualize and normalize proteins on a gel or on a membrane (post-transfer). The No-Stain reagent can covalently bind to all proteins on gels or membranes within 10 minutes, does not require any de-staining steps, and can be instantly visualized using any UV, blue light, or fluorescence imagers. The No-Stain reagent does not require any special gels or reagents and is compatible with existing gel staining and western blotting workflows. We present data and analysis demonstrating the ease of use and improved accuracy and reproducibility of quantitative western blot data with No-Stain Protein Labeling Reagent compared to housekeeping proteins.