采用蓝色非变性聚丙烯酰胺凝胶电泳和抗体凝胶迁移实验评估Bak和Bax的构象变化及寡聚化。
Blue Native PAGE and Antibody Gel Shift to Assess Bak and Bax Conformation Change and Oligomerization.
作者信息
Dewson Grant
机构信息
Cell Signalling and Cell Death Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Melbourne, Victoria 3052, Australia;Department of Medical Biology, The University of Melbourne, Melbourne, Victoria 3010, Australia.
出版信息
Cold Spring Harb Protoc. 2015 May 1;2015(5):485-9. doi: 10.1101/pdb.prot086488.
Blue native PAGE (BN-PAGE) uses Coomassie dye rather than denaturing SDS to provide a negative charge to proteins for electrophoresis. As such, it is a useful assay for investigating native supramolecular membrane complexes without the need for cross-linking. As Bak and Bax oligomers form in the mitochondrial outer membrane, and they can be efficiently monitored by BN-PAGE. Furthermore, BN-PAGE performed in conjunction with gel-shift using conformation-specific antibodies can provide additional information regarding the activation state of Bak or Bax in specific membrane complexes.
蓝色天然聚丙烯酰胺凝胶电泳（BN-PAGE）使用考马斯染料而非变性的十二烷基硫酸钠（SDS）来为蛋白质提供负电荷以进行电泳。因此，它是一种用于研究天然超分子膜复合物而无需交联的有用检测方法。由于Bak和Bax寡聚体在线粒体外膜中形成，并且可以通过BN-PAGE有效地监测它们。此外，结合使用构象特异性抗体的凝胶迁移进行的BN-PAGE可以提供有关特定膜复合物中Bak或Bax激活状态的额外信息。
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