Reversible Ponceau staining as a loading control alternative to actin in Western blots
Abstract
It is becoming standard practice to measure a housekeeping gene, typically actin, in Western blots, as it is the rule in RNA blots. We have applied reversible Ponceau staining to check equal loading of gels and measured actin in parallel under different conditions. Our results show that densitometric analysis is comparable with both techniques. Therefore, routine quantitation of Ponceau staining before antibody probing is validated as an alternative to actin blotting.
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To adjust for protein loading differences, bands of interest were normalized to either β-actin or Ponceau protein stain performed on the same blot.
“To adjust for protein loading differences, bands of interest were normalized to either β-actin or Ponceau protein stain (low-high molecular weight range included) performed on the same blot [37].”
From Synthesis of IL-6 by Hepatocytes Is a Normal Response to Common Hepatic Stimuli (2014) · cites this paper as [37]
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- Authors
- Romero-Calvo, I., Ocón, B., Martínez-Moya, P., Suárez, M. D., Zarzuelo, A., Martínez-Augustin, O., & de Medina, F. S.
- Affiliations
- Universidad de Granada (ES)
- Venue
- Analytical Biochemistry
- Publisher
- Elsevier BV
- Published
- 2010-06
- Volume
- 401
- Issue
- 2
- Pages
- 318-320
- ISSN
- 0003-2697 (ISSN-L)
- OpenAlex
- W2081680109
- Cited by
- 771
- References
- 1
- Citations by year
- 2026: 16 · 2025: 26 · 2024: 34 · 2023: 44 · 2022: 45 · 2021: 43 · 2020: 52 · 2019: 58 · 2018: 76 · 2017: 59 · 2016: 60 · 2015: 56 · 2014: 58 · 2013: 68 · 2012: 43
- FWCI
- 18.49
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Analytical Biochemistry (publishedVersion)
- PubMed (publishedVersion)
- Funders
- the Ministerio de Ciencia e Innovación
- the Instituto de Salud Carlos III
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
- Keywords
- loading control, actin, Western blot, densitometric analysis, protein quantification, housekeeping genes
- MeSH terms
- Actins, Animals, Cell Line, Colon, Humans, Kidney, Liver, Staining and Labeling, Blotting, Western, Mice, Rats
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