The Molecular Psychiatry Laboratory, the Florey Institute for Neuroscience and Mental Health, Parkville, Victoria, Australia.
The Cooperative Research Centre for Mental Health, Parkville, Victoria, Australia.
Electrophoresis. 2019 Jan;40(2):247-253. doi: 10.1002/elps.201800328. Epub 2018 Nov 6.
Levels of a reference protein must be the same as a proportion of total protein in all tissues and, in the study of human diseases, cannot vary with factors such as age, gender or disease pathophysiology. It is increasingly apparent that there may be few, if any, proteins that display the characteristics of a reference protein within the human central nervous system (CNS). To begin to challenge this hypothesis, we used Western blotting to compare variance in levels of the "gold standard" reference protein, β-actin, in Brodmann's area 9 from 194 subjects to variance of total transferred protein measured as intensity of Ponceau S staining. The coefficient of variance of sum intensity measurements for β-actin levels across all donors was 47% compared to 24 and 27% for the sum intensity of Ponceau S staining measured using two different detection techniques. These data strongly suggest that the level of β-actin, proportional to total protein, is not constant in human cortex which raises further doubt about the use of reference proteins to normalise data in human CNS studies. Considering our data, we suggest an alternative approach to presenting data from Western blotting of human CNS.
参考蛋白的水平必须与所有组织中的总蛋白比例相同，在人类疾病的研究中，不能因年龄、性别或疾病病理生理学等因素而有所差异。越来越明显的是，在人类中枢神经系统（CNS）中，可能很少有（如果有的话）蛋白质表现出参考蛋白的特征。为了开始挑战这一假设，我们使用 Western blot 比较了来自 194 个供体的布罗德曼 9 区中“黄金标准”参考蛋白β-肌动蛋白水平的差异，与使用两种不同检测技术测量的 Ponceau S 染色强度表示的总转移蛋白的差异。所有供体的β-肌动蛋白水平总和强度测量的变异系数为 47%，而使用两种不同检测技术测量的 Ponceau S 染色强度总和强度测量的变异系数为 24%和 27%。这些数据强烈表明，与总蛋白成比例的β-肌动蛋白水平在人类皮质中并不恒定，这进一步怀疑在人类中枢神经系统研究中使用参考蛋白来归一化数据的合理性。考虑到我们的数据，我们建议对人类中枢神经系统 Western blot 数据提出一种替代的呈现方法。
