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

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 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.

Methods for Peptide and Protein Quantitation by Liquid Chromatography-Multiple Reaction Monitoring Mass Spectrometry

“…The LRP and LF methods are well suited to estimate differences in expression for a few proteins in a small number of samples, as is frequently done in biochemistry and cell biology studies. These analyses typically do not require high precision and are done by immunoblotting, which yields CVs in the range from 20 -40% (45,46). LRP is well-suited to screen biomarker candidate proteins in larger collections of biospecimens corresponding to multiple phenotypes.…”

Transparency Is the Key to Quality

“…This is further complicated by the fact that some detection methods, in particular enhanced chemiluminescence using x-ray film, have a very restricted linear range, and careful attention to the experimental conditions is necessary to ensure linearity. It is typically better to normalize Western blots using total protein loading as the denominator (3)(4)(5)(6)(7)(8)(9)(10)(11). To avoid potential pitfalls and with a focus on improving transparency, the JBC strongly recommends that authors describe their methods used to quantify signal intensity, how the linearity of signal intensity with antigen loading was established, and how protein loading was normalized between lanes.…”

Chronic Intermittent Ethanol Exposure Enhances the Excitability and Synaptic Plasticity of Lateral Orbitofrontal Cortex Neurons and Induces a Tolerance to the Acute Inhibitory Actions of Ethanol

“…The bands were quantified by mean optical density using computer-assisted densitometry with ImageJ v1.41 (National Institutes of Health, USA). As loading controls (eg, actin and GAPDH) often used for in western blot experiments are subject to quantitation errors (Aldridge et al, 2008;Dittmer and Dittmer, 2006), each gel was normalized to a total protein stain (Swift stain; G-Biosciences, St Louis, MO). Before each study, a series of western blottings were performed using different titrations of sample and antibody, to establish the linear range for each response.…”