Biological validation of a novel process and product for quantitating western blots

Abstract

Protein normalization of western blots has relied upon housekeeping proteins which exhibit signal saturation and varied cellular expression level variations. These issues can produce spurious results leading to erroneous conclusions. A superior method to protein normalization using housekeeping proteins is Total Protein Normalization, a method now recognized as the gold standard for quantitative westerns. Total Protein Normalization requires that all proteins on a membrane be stained or labeled uniformly, imaged, and then analyzed for total protein. It is important that such a normalization process not interfere with typical immunodetection methods, fits within existing western workflows, and exhibits a linear relationship of signal intensity to protein load under all experimental conditions. Here we report that we developed a new reagent enabling Total Protein Normalization, and we demonstrate its superior protein normalization capabilities through analysis of target proteins in different cell backgrounds. These data illustrate how housekeeping proteins exhibit signal saturation, yield erroneous normalization data, and display sample-to-sample variations averaging 48.2 % overall. Signal intensities obtained using our new method show a linear relationship to protein sample load, thus providing accurate protein normalization with an overall average variation of 7.7 %.

Keywords: Housekeeping proteins; Quantitative westerns; Total protein normalization; Western blots.