The use of total protein stains as loading controls: An alternative to high-abundance single-protein controls in semi-quantitative immunoblotting

Georgina M Aldridge; David M Podrebarac; William T Greenough; Ivan Jeanne Weiler

doi:10.1016/j.jneumeth.2008.05.003

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The use of total protein stains as loading controls: An alternative to high-abundance single-protein controls in semi-quantitative immunoblotting

Journal article

Peer reviewed

The use of total protein stains as loading controls: An alternative to high-abundance single-protein controls in semi-quantitative immunoblotting

Georgina M Aldridge, David M Podrebarac, William T Greenough and Ivan Jeanne Weiler

Journal of neuroscience methods, Vol.172(2), pp.250-254

07/30/2008

DOI: 10.1016/j.jneumeth.2008.05.003

PMID: 18571732

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Abstract

Western blots are used to estimate the relative concentrations of proteins of interest based on staining by specific antibodies. Quantitative measurements are often subject to error due to overloading of the loading control and over-reliance on normalization. We have found that at the protein concentrations normally used to quantify most low-abundance proteins of interest, frequently used single-protein loading controls, such as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-actin, do not accurately reflect differences in protein concentration. Two total protein stains, SYPRO® Ruby and Amido Black, were compared and found to be acceptable alternatives to single-protein controls. Although we cannot prove that high-abundance loading controls are inaccurate under all possible conditions, we conclude that the burden of proof should lie with the researcher to demonstrate that their loading control is reflective of quantitative differences in protein concentration.

Western blot

Loading control

Immunoblot

Normalization

Details

Title: Subtitle: The use of total protein stains as loading controls: An alternative to high-abundance single-protein controls in semi-quantitative immunoblotting
Creators: Georgina M Aldridge - Beckman Institute, University of Illinois at Urbana-Champaign, USA
David M Podrebarac - Beckman Institute, University of Illinois at Urbana-Champaign, USA
William T Greenough - Beckman Institute, University of Illinois at Urbana-Champaign, USA
Ivan Jeanne Weiler - Beckman Institute, University of Illinois at Urbana-Champaign, USA
Resource Type: Journal article
Publication Details: Journal of neuroscience methods, Vol.172(2), pp.250-254
DOI: 10.1016/j.jneumeth.2008.05.003
PMID: 18571732
NLM abbreviation: J Neurosci Methods
ISSN: 0165-0270
eISSN: 1872-678X
Publisher: Elsevier B.V
Language: English
Date published: 07/30/2008
Academic Unit: Neurology; Iowa Neuroscience Institute
Record Identifier: 9984018134202771

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