Evaluation of SYPRO Ruby total protein stain for the normalization of two-dimensional Western blots

Birgit Steinberger; Gottfried Brem; Corina Mayrhofer

doi:10.1016/j.ab.2015.01.015

Evaluation of SYPRO Ruby total protein stain for the normalization of two-dimensional Western blots

Anal Biochem. 2015 May 1:476:17-9. doi: 10.1016/j.ab.2015.01.015. Epub 2015 Jan 30.

Authors

Birgit Steinberger¹, Gottfried Brem², Corina Mayrhofer³

Affiliations

¹ Institute of Animal Breeding and Genetics, Department of Biomedical Sciences, University of Veterinary Medicine, A-1210 Vienna, Austria; Institute of Biotechnology in Animal Production, Department of Agrobiotechnology (IFA Tulln), University of Natural Resources and Applied Life Sciences Vienna, A-3430 Tulln, Austria. Electronic address: birgit.steinberger@boku.ac.at.
² Institute of Animal Breeding and Genetics, Department of Biomedical Sciences, University of Veterinary Medicine, A-1210 Vienna, Austria.
³ Institute of Animal Breeding and Genetics, Department of Biomedical Sciences, University of Veterinary Medicine, A-1210 Vienna, Austria; Institute of Biotechnology in Animal Production, Department of Agrobiotechnology (IFA Tulln), University of Natural Resources and Applied Life Sciences Vienna, A-3430 Tulln, Austria.

PMID: 25640586
DOI: 10.1016/j.ab.2015.01.015

Abstract

Due to post-translational modifications such as phosphorylation, proteins exist as distinct charge variants. Two-dimensional (2D) gel electrophoresis followed by immunoblotting enables the detection of these isoforms. For their accurate relative quantitation in different samples, a loading control is necessary to compensate for technical errors such as imprecise sample loading or transfer. The study reveals that the combinatory approach of SYPRO Ruby and chemiluminescence-based 2D Western blot analysis exhibits high linearity and excellent reproducibility and is applicable for limited sample amounts.

Keywords: Isoform quantitation; Normalization; SYPRO Ruby; Two-dimensional Western blot.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Blotting, Western
Electrophoresis, Gel, Two-Dimensional / methods*
Organometallic Compounds / chemistry*

Substances

Organometallic Compounds
Sypro Ruby