The use of total protein stains as loading controls: An alternative to high-abundance single-protein controls in semi-quantitative immunoblotting
The use of total protein stains as loading controls: An alternative to high-abundance single-protein controls in semi-quantitative immunoblotting
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DOI：
10.1016/j.jneumeth.2008.05.003
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发表时间：
2008-07-30
影响因子：
3
通讯作者：
Weiler, Ivan Jeanne
中科院分区：
文献类型：
--
作者：
Aldridge, Georgina M.;Podrebarac, David M.;Weiler, Ivan Jeanne
Western blotts用于基于特定抗体的染色来估计感兴趣蛋白质的相对浓度。由于加载控制过载和过度依赖归一化，定量测量经常会出现误差。我们发现，在通常用于定量大多数低丰度蛋白质的蛋白质浓度下，常用的单一蛋白质负荷对照，如甘油醛3-磷酸脱氢酶(GAPDH)和P-肌动蛋白，不能准确反映蛋白质浓度的差异。两种全蛋白染料SYPRO(R)Ruby和Amido Black进行了比较，发现它们是单蛋白对照的可接受替代品。虽然我们不能证明高丰度负荷控制在所有可能的条件下都是不准确的，但我们的结论是，举证责任应由研究人员承担，以证明其负荷控制反映了蛋白质浓度的数量差异(C)2008 Elsevier B.V.保留所有权利。
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.