Biochemistry and Biophysics Graduate Program, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA; Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA.
Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA; Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA.
Anal Biochem. 2019 Apr 15;571:21-24. doi: 10.1016/j.ab.2019.02.008. Epub 2019 Feb 16.
Sodium dodecyl sulfate (SDS) is a detergent used as a strong denaturant of proteins in gel electrophoresis. It has previously been shown that certain hyperstable, also known as kinetically stable, proteins are resistant to SDS and thus require heating for their denaturation in the presence of SDS. Because of its high denaturing strength, relatively few proteins are resistant to SDS thereby limiting the current use of SDS-PAGE for identifying hyperstable degradation-resistant proteins. In this study, we show that sarkosyl, a milder detergent than SDS, is able to identify proteins with moderately high kinetic stability that lack SDS-resistance. Our assay involves running and subsequently comparing boiled and unheated protein samples containing sarkosyl, instead of SDS, on PAGE gels and identifying subsequent differences in protein migration. Our results also show that sarkosyl and SDS may be combined in PAGE experiments at varying relative percentages to obtain semi-quantitative information about a protein's kinetic stability in a range inaccessible by probing through native- or SDS-PAGE. Using protein extracts from various legumes as model systems, we detected proteins with a range of protein stability from nearly SDS-resistant to barely sarkosyl resistant.
十二烷基硫酸钠（SDS）是一种在凝胶电泳中用作蛋白质强烈变性剂的洗涤剂。先前已经表明，某些超稳定的蛋白质，也称为动力学稳定的蛋白质，对 SDS 具有抗性，因此在 SDS 存在下需要加热使其变性。由于 SDS 的高变性强度，相对较少的蛋白质对 SDS 具有抗性，从而限制了 SDS-PAGE 当前用于鉴定超稳定抗降解蛋白的用途。在这项研究中，我们表明，Sarkosyl 是一种比 SDS 更温和的洗涤剂，能够鉴定出具有中度高动力学稳定性但缺乏 SDS 抗性的蛋白质。我们的测定法包括在含有 Sarkosyl 而不是 SDS 的 PAGE 凝胶上运行和随后比较煮沸和未加热的蛋白质样品，并鉴定蛋白质迁移的后续差异。我们的结果还表明，Sarkosyl 和 SDS 可以在 PAGE 实验中以不同的相对百分比组合使用，以获得通过探测天然或 SDS-PAGE 无法获得的范围内有关蛋白质动力学稳定性的半定量信息。使用来自各种豆科植物的蛋白质提取物作为模型系统，我们检测到具有从几乎对 SDS 具有抗性到几乎对 Sarkosyl 具有抗性的蛋白质稳定性范围的蛋白质。
