Hurst Charlotte H, Turnbull Dionne, Hemsley Piers A
Division of Plant Sciences, School of Life Sciences, University of Dundee, Dundee, Scotland, UK.
Cell and Molecular Sciences, The James Hutton Institute, Dundee, Scotland, UK.
Methods Mol Biol. 2019;2009:3-11. doi: 10.1007/978-1-4939-9532-5_1.
S-Acylation is increasingly being recognized as an important dynamic posttranslational modification of cysteine residues in proteins. Various approaches have been described for assaying protein S-acylation with acyl-switch approaches being the most common and accessible. However, these approaches can be time-consuming with low reproducibility as a result of multiple protein precipitation/resuspension cleanup steps. Here we present a faster, cleaner, and more sensitive acyl-switch approach for detecting the S-acylation state of any protein, from any cell or tissue type, that can be detected by western blotting. In the case of acyl-RAC, the procedure is now performed without protein precipitation, greatly increasing speed and improving sample handling in the assay. This also allows for more samples to be processed simultaneously and opens the way for medium-throughput assays. Overall, maleimide scavenging improves the reliability of determination and quantification of protein S-acylation state by acyl-switch methods.
蛋白质的S-酰化越来越被认为是蛋白质中半胱氨酸残基的一种重要的动态翻译后修饰。已经描述了多种用于检测蛋白质S-酰化的方法，其中酰基转换方法是最常见且易于操作的。然而，由于多个蛋白质沉淀/重悬纯化步骤，这些方法可能耗时且重现性低。在此，我们提出了一种更快、更简便且更灵敏的酰基转换方法，用于检测任何细胞或组织类型中任何可通过蛋白质印迹法检测的蛋白质的S-酰化状态。对于酰基-RAC，现在该程序无需蛋白质沉淀即可进行，大大提高了速度并改善了检测中的样品处理。这也允许同时处理更多样品，并为中等通量检测开辟了道路。总体而言，马来酰亚胺清除提高了通过酰基转换方法测定和定量蛋白质S-酰化状态的可靠性。
