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Protein quantification and its tolerance for different interfering reagents using the BCA-method with regard to 2D SDS PAGE
Abstract
Measuring the protein content of a sample is a mandatory and frequently practiced procedure in the lab. Although the procedure is quite simple and convenient to perform with commercially available kits, incompatible reagents in the lysate can cause problems in the quality of measurement. Unfortunately these reagents are cornerstones of high efficiency lysing buffers, e.g. high amounts of urea or beta-mercaptoethanol. In this study we addressed the tolerance of the well-known BCA-assay (bicinchoninic acid) to various reagents in different concentrations, with special regard to a subsequent 2D-gelelectrophoresis. As a result, the kit is incompatible with the recipes of regular 2D-buffers. Also, when mixing two different reagents interfering effects will occur in a non-predictable way. Therefore we established a new method to quantify protein content in lysates ready for 2D-gelelectrophoresis: by mixing an aliquot with SDS, an equilibration is performed to that the sample can be run on a regular 1D SDS PAGE. Image analysis following fluorescence staining (SYPRO Ruby) reveals the absolute protein content in comparison to a BSA dilution curve processed accordingly.
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“The methodology was validated considering the tolerance criteria described by Krieg et al. [16].”
From Functional and Bioactive Characterization of Hemp Cake Proteins and Polyphenols from Non-Psychoactive Cannabis sativa (2025) · cites this paper as Krieg et al. [16]
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Metadata
- Authors
- Krieg, R. C., Dong, Y., Schwamborn, K., & Knuechel, R.
- Affiliations
- RWTH Aachen University (DE)
- Venue
- Journal of Biochemical and Biophysical Methods
- Publisher
- Elsevier BV
- Published
- 2005-10
- Volume
- 65
- Issue
- 1
- Pages
- 13-19
- ISSN
- 0165-022X (ISSN-L)
- OpenAlex
- W2068769109
- Cited by
- 122
- References
- 6
- Citations by year
- 2026: 7 · 2025: 6 · 2024: 6 · 2023: 9 · 2022: 9 · 2021: 5 · 2020: 6 · 2019: 6 · 2018: 9 · 2017: 11 · 2016: 6 · 2015: 12 · 2014: 7 · 2013: 3 · 2012: 1
- FWCI
- 0.86
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Journal of Biochemical and Biophysical Methods (publishedVersion)
- PubMed (publishedVersion)
- RWTH Publications (RWTH Aachen) (submittedVersion)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Advanced Biosensing Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
- Keywords
- protein quantification, bicinchoninic acid assay, urea, 2-mercaptoethanol
- MeSH terms
- Proteins, Quinolines, Reagent Kits, Diagnostic, Sensitivity and Specificity, Electrophoresis, Gel, Two-Dimensional, Reproducibility of Results
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