文章基本信息

标题：Validating Antibodies for Quantitative Western Blot Measurements with Microwestern Array
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作者：Rick J. Koch ; Anne Marie Barrette ; Alan D. Stern 等
期刊名称：Scientific Reports
电子版ISSN：2045-2322
出版年度：2018
卷号：8
期号：1
页码：11329
DOI：10.1038/s41598-018-29436-0
语种：English
出版社：Springer Nature
摘要：Fluorescence-based western blots are quantitative in principal, but require determining linear range for each antibody. Here, we use microwestern array to rapidly evaluate suitable conditions for quantitative western blotting, with up to 192 antibody/dilution/replicate combinations on a single standard size gel with a seven-point, two-fold lysate dilution series (~100-fold range). Pilot experiments demonstrate a high proportion of investigated antibodies (17/24) are suitable for quantitative use; however this sample of antibodies is not yet comprehensive across companies, molecular weights, and other important antibody properties, so the ubiquity of this property cannot yet be determined. In some cases microwestern struggled with higher molecular weight membrane proteins, so the technique may not be uniformly applicable to all validation tasks. Linear range for all validated antibodies is at least 8-fold, and up to two orders of magnitude. Phospho-specific and total antibodies do not have discernable trend differences in linear range or limit of detection. Total antibodies generally required higher working concentrations, but more comprehensive antibody panels are required to better establish whether this trend is general or not. Importantly, we demonstrate that results from microwestern analyses scale to normal "macro" western for a subset of antibodies.