期刊
JOURNAL OF NEUROSCIENCE METHODS
卷 172, 期 2, 页码 250-254出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jneumeth.2008.05.003
关键词
western blot; loading control; immunoblot normalization
资金
- NICHD NIH HHS [T32 HD007333, T32 HD007333-22, HD007333] Funding Source: Medline
- NIMH NIH HHS [R01 MH035321-22, R37 MH035321, R01 MH035321, MH35321] Funding Source: Medline
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 P-actin, do not accurately reflect differences in protein concentration. Two total protein stains, SYPRO (R) 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 (c) 2008 Elsevier B.V. All rights reserved.
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