The use of total protein stains as loading controls: an alternative to high-abundance single-protein controls in semi-quantitative immunoblotting.
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 beta-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.
Aldridge
Georgina M
GM
Beckman Institute, University of Illinois at Urbana-Champaign, USA. galdri2@uiuc.edu
Podrebarac
David M
DM
Greenough
William T
WT
Weiler
Ivan Jeanne
IJ
D000580:Amido Black / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D001923:Brain Chemistry / Q000502:physiology; D004396:Coloring Agents / Q000737:chemistry; D000076122:Disks Large Homolog 4 Protein; D051528:Guanylate Kinases; D047908:Intracellular Signaling Peptides and Proteins / Q000032:analysis / Q000737:chemistry; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry; D051379:Mice; D048052:Mitogen-Activated Protein Kinase 3 / Q000032:analysis / Q000737:chemistry; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000737:chemistry*; D009448:Neurochemistry / Q000379:methods*; D009942:Organometallic Compounds / Q000737:chemistry; D040901:Proteomics; D013194:Staining and Labeling / Q000379:methods*; D013347:Subcellular Fractions
D004396:Coloring Agents; D000076122:Disks Large Homolog 4 Protein; C501447:Dlg4 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D009942:Organometallic Compounds; C515601:Sypro Ruby; D048052:Mitogen-Activated Protein Kinase 3; D051528:Guanylate Kinases; D000580:Amido Black