Ponceau S waste: Ponceau S staining for total protein normalization

• Published in: Analytical biochemistry 2019-06, Vol.575, p.44-53
• Main Authors: Sander, Hannah, Wallace, Samantha, Plouse, Rachel, Tiwari, Shuchita, Gomes, Aldrin V.
• Format: Article
• Language: English
• Subjects: acetic acid; Azo Compounds - chemistry; Blotting, Western; Coloring Agents - chemistry; gels; Internet; Ponceau S; protein content; Proteins - chemistry; staining; Staining and Labeling; Total protein staining; trichloroacetic acid; wastes; Western blot normalization; Western blotting
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2019.03.010

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. •Total protein staining offers several advantages for Western blotting normalization.•Ponceau S is the most common total protein stain for Western blotting normalization.•Most laboratories are using excessive amounts of Ponceau S and different acids.•0.01% (w/v) Ponceau S in 1% acetic acid is as effective as all other formulations.•Only 1–2 min of staining is required for total protein staining with Ponceau S.