Abstract
Determination of protein concentrations prior to (sodium dodecyl sulfate) SDS electrophoresis is made difficult by the simultaneous presence of SDS and reducers in the buffers used for protein extraction. Reducers interfere with the copper-based assays, while SDS interferes with the dye-binding assays. The combined use of cyclodextrins with a commercial Bradford reagent concentrate, described in this chapter, allows to determine protein concentrations in a Laemmli-type buffer, containing both SDS and reducers, in a single step (without any precipitation) with a simple spectrophotometric assay. The use of various cyclodextrins brings compatibility not only with SDS but also with other nonionic and ionic detergents such as sodium deoxycholate or detergents of the Triton type.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Schirle M, Heurtier MA, Kuster B (2003) Profiling core proteomes of human cell lines by one-dimensional PAGE and liquid chromatography-tandem mass spectrometry. Mol Cell Proteomics 2:1297–1305
Rabilloud T (2012) The whereabouts of 2D gels in quantitative proteomics. Methods Mol Biol 893:25–35
Welinder C, Ekblad L (2011) Coomassie staining as loading control in western blot analysis. J Proteome Res 10:1416–1419
Eaton SL, Roche SL, Llavero Hurtado M, Oldknow KJ et al (2013) Total protein analysis as a reliable loading control for quantitative fluorescent western blotting. PLoS One 8:e72457
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Smith PK, Krohn RI, Hermanson GT, Mallia AK et al (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Fujita Y, Mori I, Kitano S (1984) Determination of proteins by using the color reaction with pyrocatechol violet-molybdenum(VI) complex. Chem Pharm Bull (Tokyo) 32:4161–4164
Bensadoun A, Weinstein D (1976) Assay of proteins in the presence of interfering materials. Anal Biochem 70:241–250
Peterson GL (1977) A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem 83:346–356
Rabilloud T (2016) A single step protein assay that is both detergent and reducer compatible: the cydex blue assay. Electrophoresis 37:2595–2601
Neuhoff V, Arold N, Taube D, Ehrhardt W (1988) Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie brilliant blue G-250 and R-250. Electrophoresis 9:255–262
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Rabilloud, T. (2019). The Cydex Blue Assay: A One-Step Protein Assay for Samples Prior to SDS Electrophoresis. In: Kurien, B., Scofield, R. (eds) Electrophoretic Separation of Proteins. Methods in Molecular Biology, vol 1855. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8793-1_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8793-1_6
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8792-4
Online ISBN: 978-1-4939-8793-1
eBook Packages: Springer Protocols