Ponceau S waste: Ponceau S staining for total protein normalization

Anal Biochem. 2019 Jun 15:575:44-53. doi: 10.1016/j.ab.2019.03.010. Epub 2019 Mar 23.


Normalization of Western blotting data is a critical step that is needed to reduce errors caused by unequal sample loading across lanes in a gel, inconsistent sample preparation, and variations due to experimental errors. Several papers have suggested that total protein normalization may be better than housekeeping protein normalization for Western blotting normalization. Ponceau S is the most commonly used stain for total protein normalization. A review of the literature and commercial websites suggest a multitude of Ponceau S staining protocols for total protein staining of blots. In this study, we explored which Ponceau S staining protocol would result in the highest sensitivity of protein band detection. Unexpectedly, we found that irrespective of the Ponceau S concentration (between 0.001 and 2% (w/v)), acid concentration, and acid type (acetic acid, trichloroacetic acid and/or sulfosalicylic acid), the sensitivity of protein detection remained constant. The most commonly used concentration of Ponceau S is 0.1%, while 0.001% (100-fold less) Ponceau S resulted in the same sensitivity of protein band detection. We suggest the use of the relatively inexpensive 0.01% Ponceau S in 1% acetic acid stain for total protein normalization as it is as effective as all the expensive formulations that are currently used.

Keywords: Ponceau S; Total protein staining; Western blot normalization.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Azo Compounds / chemistry*
  • Blotting, Western
  • Coloring Agents / chemistry*
  • Proteins / chemistry*
  • Staining and Labeling*


  • Azo Compounds
  • Coloring Agents
  • Proteins
  • ponceau S