Total protein or high-abundance protein: Which offers the best loading control for Western blotting?
Total protein or high-abundance protein: Which offers the best loading control for Western blotting?
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DOI：
10.1016/j.ab.2015.11.022
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发表时间：
2016-03-01
影响因子：
2.9
通讯作者：
Mielke, John G.
中科院分区：
文献类型：
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作者：
Thacker, Jonathan S.;Yeung, Derrick H.;Mielke, John G.
蛋白质印迹法通常涉及对免疫印迹泳道中蛋白质量的可变性的控制。将靶信号标准化为针对大量表达的蛋白质发现的信号被广泛认为是可靠的上样控制;然而，这种方法正受到越来越多的质疑。因此，我们比较了两种高丰度蛋白质（肌动蛋白和甘油醛3-磷酸脱氢酶[GAPDH]）和两种总蛋白膜染色方法（丽春红和考马斯亮蓝）的印迹，以确定最佳的控制上样变异性。我们发现，丽春红染色最佳平衡的准确性和精度，我们建议，这种方法被认为是一种替代标准化与高丰度蛋白质。(c)2015 Elsevier Inc. All rights reserved.
Western blotting routinely involves a control for variability in the amount of protein across immunoblot lanes. Normalizing a target signal to one found for an abundantly expressed protein is widely regarded as a reliable loading control; however, this approach is being increasingly questioned. As a result, we compared blotting for two high-abundance proteins (actin and glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) and two total protein membrane staining methods (Ponceau and Coomassie Brilliant Blue) to determine the best control for loading variability. We found that Ponceau staining optimally balanced accuracy and precision, and we suggest that this approach be considered as an alternative to normalizing with a high-abundance protein. (c) 2015 Elsevier Inc. All rights reserved.