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

• Published in: Journal of neuroscience methods Vol. 172; no. 2; pp. 250 - 254
• Main Authors: Aldridge, Georgina M., Podrebarac, David M., Greenough, William T., Weiler, Ivan Jeanne
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 30.07.2008
• Subjects: Amido Black - chemistry; Amino Acid Sequence; Animals; Blotting, Western - methods; Brain Chemistry - physiology; Coloring Agents - chemistry; Disks Large Homolog 4 Protein; Guanylate Kinases; Immunoblot; Intracellular Signaling Peptides and Proteins - analysis; Intracellular Signaling Peptides and Proteins - chemistry; Loading control; Membrane Proteins - analysis; Membrane Proteins - chemistry; Mice; Mitogen-Activated Protein Kinase 3 - analysis; Mitogen-Activated Protein Kinase 3 - chemistry; Nerve Tissue Proteins - analysis; Nerve Tissue Proteins - chemistry; Neurochemistry - methods; Normalization; Organometallic Compounds - chemistry; Proteomics; Staining and Labeling - methods; Subcellular Fractions; Western blot; Western blot; Loading control; Immunoblot; Normalization
• ISSN: 0165-0270; 1872-678X
• DOI: 10.1016/j.jneumeth.2008.05.003

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.