Division of Molecular Structure and Function, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8.
Anal Biochem. 2013 Mar 1;434(1):67-72. doi: 10.1016/j.ab.2012.11.007. Epub 2012 Nov 29.
Membrane proteins are known to migrate anomalously on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to relative molecular mass (M(r)) values larger or smaller than formula molecular weight. We constructed a database from literature M(r) values reported for 168 nonredundant helical membrane proteins with structures determined to high resolution and found that more than three-quarters of them exhibit this behavior on gels calibrated with commercial standards. Further analysis of the database indicated that the direction of anomalous migration is not a consequence of membrane protein net charge or hydrophobicity. Plots of observed versus formula M(r) values showed that membrane proteins migrating slower than expected read out at 1.13×M(r), whereas those that migrate faster than expected read out at 0.82×M(r) (R(2)~0.98, P<0.0001). These robust trends imply that division of the M(r) readouts of slower or faster migrating analytes by 1.13 or 0.82, respectively, should enhance SDS-PAGE accuracy. Applying this correction procedure to SDS-PAGE readouts of four fast-migrating helical transmembrane (TM) proteins significantly reduced M(r) errors from approximately 20% to 8% (P<0.0001). Our results suggest that hydrophobic standards for SDS-PAGE would significantly improve the performance of the technique applied to membrane proteins.
膜蛋白在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）上的迁移通常会出现异常，导致其相对分子质量（Mr）值大于或小于理论分子量。我们从文献中构建了一个数据库，其中包含 168 种非冗余螺旋膜蛋白的 Mr 值，这些蛋白的结构已被确定为高分辨率结构，并发现其中超过四分之三的蛋白在使用商业标准校准的凝胶上表现出这种异常迁移行为。对数据库的进一步分析表明，异常迁移的方向不是膜蛋白净电荷或疏水性的结果。观察到的 Mr 值与理论 Mr 值的关系图表明，迁移速度比预期慢的膜蛋白的读数为 1.13×Mr，而迁移速度比预期快的膜蛋白的读数为 0.82×Mr（R2~0.98，P<0.0001）。这些稳健的趋势表明，分别将较慢或较快迁移分析物的 Mr 读数除以 1.13 或 0.82，应该可以提高 SDS-PAGE 的准确性。将该校正程序应用于四个快速迁移的螺旋跨膜（TM）蛋白的 SDS-PAGE 读数，显著降低了 Mr 误差，从大约 20%降低到 8%（P<0.0001）。我们的结果表明，SDS-PAGE 的疏水性标准将显著提高该技术应用于膜蛋白的性能。
