A fluorescent Ponceau S-based total protein normalization method for conventional and challenging immunoblot samples.

Immunoblotting normalization issues have been recently overcome by whole lane staining. Herein, we are taking advantage of these recent advances and of the fluorophore status of the Ponceau S stain in order to combine the advantages of whole lane staining and fluorescence. By Ponceau S excitation at 488 nm, we identify the so-called 'fluorescent Ponceau' method as more linear, more sensitive and more repeatable than the others in protein lysates of distant biochemical profiles (cells, human and mouse tissues). This essentially cost-free method at the single experiment level provides accessible and robust means for post-blot normalization of many types of analytes. [Display omitted] • 'Housekeeping protein' signals robustness is much problematic in western blotting. • Ponceau Red is a fluorescent dye, amenable to fluorescence-based quantification. • Herein, a novel fluorescent Ponceau-based normalization technique is detailed. • It outcompetes regular Ponceau and pertinent commercial products. • Criteria used were sensitivity, linearity, and repeatability. [ABSTRACT FROM AUTHOR]