Mollica Janelle P, Oakhill Jonathan S, Lamb Graham D, Murphy Robyn M
Department of Zoology, La Trobe University, Melbourne, Vic. 3086, Australia.
Anal Biochem. 2009 Mar 15;386(2):270-5. doi: 10.1016/j.ab.2008.12.029. Epub 2009 Jan 4.
This study used purified calsequestrin 1 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 nonproportional, standard curve resulted in approximately fourfold error. The standard curve method was used to estimate the concentration of AMPK beta1 in rat extensor digitorum longus muscle as being of the order of 60 microM. The article suggests that adopting a more sensitive Western blotting protocol will improve the reliability of quantitative Western blotting outcomes.
本研究使用纯化的肌集钙蛋白1和AMP激酶（AMPK）蛋白，以证明当检测到的蛋白质密度处于标准曲线的非比例区域内，或者在数据分析中未考虑标准曲线时，蛋白质印迹结果会受到怎样的影响。它概述了仅使用条带密度和/或分析过多样品这一简单错误导致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（尽管是非比例的）标准曲线进行外推，结果产生了约四倍的误差。使用标准曲线法估计大鼠趾长伸肌中AMPK β1的浓度约为60微摩尔。该文章表明，采用更灵敏的蛋白质印迹方案将提高定量蛋白质印迹结果的可靠性。
