Ponceau S waste: Ponceau S staining for total protein normalization

Hannah Sander, Samantha Wallace, Rachel Plouse, Shuchita Tiwari, Aldrin V Gomes

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)44-53
Number of pages10
JournalAnalytical Biochemistry
Volume575
DOIs
StatePublished - Jun 15 2019

Fingerprint

Staining and Labeling
Proteins
Acetic Acid
Coloring Agents
Western Blotting
Network protocols
Housekeeping
Trichloroacetic Acid
Acids
ponceau S
Websites
Gels

Keywords

  • Ponceau S
  • Total protein staining
  • Western blot normalization

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Sander, H., Wallace, S., Plouse, R., Tiwari, S., & Gomes, A. V. (2019). Ponceau S waste: Ponceau S staining for total protein normalization. Analytical Biochemistry, 575, 44-53. https://doi.org/10.1016/j.ab.2019.03.010

Ponceau S waste : Ponceau S staining for total protein normalization. / Sander, Hannah; Wallace, Samantha; Plouse, Rachel; Tiwari, Shuchita; Gomes, Aldrin V.

In: Analytical Biochemistry, Vol. 575, 15.06.2019, p. 44-53.

Research output: Contribution to journalArticle

Sander, H, Wallace, S, Plouse, R, Tiwari, S & Gomes, AV 2019, 'Ponceau S waste: Ponceau S staining for total protein normalization', Analytical Biochemistry, vol. 575, pp. 44-53. https://doi.org/10.1016/j.ab.2019.03.010
Sander, Hannah ; Wallace, Samantha ; Plouse, Rachel ; Tiwari, Shuchita ; Gomes, Aldrin V. / Ponceau S waste : Ponceau S staining for total protein normalization. In: Analytical Biochemistry. 2019 ; Vol. 575. pp. 44-53.
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