Are genuine changes in protein expression being overlooked? Reassessing Western blotting
Are genuine changes in protein expression being overlooked? Reassessing Western blotting
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DOI：
10.1016/j.ab.2008.12.029
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发表时间：
2009-03-15
影响因子：
2.9
通讯作者：
Murphy, Robyn M.
中科院分区：
文献类型：
--
作者：
Mollica, Janelle P.;Oakhill, Jonathan S.;Murphy, Robyn M.
这项研究使用纯化的钙调蛋白I和AMP激酶(AMPK)蛋白来展示当检测到的蛋白质密度位于标准曲线的非比例区域内或在数据分析中没有考虑标准曲线时，蛋白质印迹结果会受到怎样的影响。它概述了通过单独使用频带密度和/或通过分析过多样本的简单错误而忽略真实变化的可能性。为了证明这一点，对一条典型的线性尽管不成比例的标准曲线进行外推，产生了大约四倍的误差。用标准曲线法估计大鼠趾长伸肌中AMPKβ1的浓度约为60mU M。本文认为采用更灵敏的Western blotting方法将提高定量Western blotting结果的可靠性。皇冠版权所有(C)2009由爱思唯尔公司出版。保留所有权利。
This study used purified calsequestrin I and AMP kinase (AMPK) proteins to demonstrate how Western blotting outcomes can be influenced when either the density of proteins detected lie within a nonproportional region of a standard curve or a standard curve is not taken into account for data analyses. It outlines the likelihood of true changes being overlooked through the simple mistake of using band density alone and/or through analyzing too much sample. To demonstrate this, extrapolation of a typical linear, although non proportional, standard curve resulted in approximately fourfold error. The standard curve method was used to estimate the concentration of AMPK beta 1 in rat extensor digitorum longus muscle as being of the order of 60 mu M. The article suggests that adopting a more sensitive Western blotting protocol will improve the reliability of quantitative Western blotting outcomes. Crown Copyright (C) 2009 Published by Elsevier Inc. All rights reserved.