Detergents play an essential role during the isolation of membrane protein complexes. Inappropriate use of detergents may affect the native fold of the membrane proteins, their binding to antibodies, or their interaction with partner proteins. Here we used cadherin-11 (Cad11) as an example to examine the impact of detergents on membrane protein complex isolation. We found that mAb 1A5 could immunoprecipitate Cad11 when membranes were solubilized by dodecyl maltoside (DDM) but not by octylglucoside, suggesting that octylglucoside interferes with Cad11-mAb 1A5 interaction. Further, we compared the effects of Brij 35, Triton X-100, cholate, CHAPSO, zwittergen 3-12, deoxyBIG CHAP, and digitonin on Cad11 solubilization and immunoprecipitation. We found that all detergents, except Brij35, could solubilize Cad11 from the membrane. Upon immunoprecipitation, we found that β-catenin, a known cadherin-interacting protein, was present in Cad11 immune complex among the detergents tested except Brij 35. However, the association of p120 catenin with Cad11 varied depending on the detergents used. Using isobaric tag for relative and absolute quantitation (iTRAQ) to determine the relative levels of proteins in Cad11 immune complexes, we found that DDM and Triton X-100 were more efficient than cholate in solubilization and immunoprecipitation of Cad11 and resulted in the identification of both canonical and new candidate Cad11-interacting proteins.

中文翻译：

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清洁剂对膜蛋白复合物分离的影响

在分离膜蛋白复合物时，洗涤剂起着至关重要的作用。洗涤剂使用不当可能会影响膜蛋白的天然折叠，与抗体的结合或与伴侣蛋白的相互作用。在这里，我们以钙黏着蛋白11（Cad11）为例，研究去污剂对膜蛋白复合物分离的影响。我们发现，当膜被十二烷基麦芽糖苷（DDM）溶解而不被辛基葡萄糖苷溶解时，mAb 1A5可以免疫沉淀Cad11，这表明辛基葡萄糖苷会干扰Cad11-mAb 1A5相互作用。此外，我们比较了Brij 35，Triton X-100，胆酸盐，CHAPSO，两性离子原3-12，deoxyBIG CHAP和洋地黄皂苷对Cad11增溶和免疫沉淀的影响。我们发现，除Brij35外，所有清洁剂都可以溶解膜中的Cad11。免疫沉淀后，我们发现除Brij 35外，在测试的去污剂中，Cad11免疫复合物中存在一种已知的钙粘蛋白相互作用蛋白β-catenin。但是，p120连环蛋白与Cad11的缔合取决于所用的去污剂。使用等压定量相对和绝对定量（iTRAQ）来确定Cad11免疫复合物中蛋白质的相对水平，我们发现DDM和Triton X-100在Cad11的增溶和免疫沉淀中比胆酸盐更有效，并导致了两者的鉴定规范和新的候选Cad11相互作用蛋白。p120连环蛋白与Cad11的缔合取决于所用的去污剂。使用等压定量相对和绝对定量（iTRAQ）来确定Cad11免疫复合物中蛋白质的相对水平，我们发现DDM和Triton X-100在Cad11的增溶和免疫沉淀中比胆酸盐更有效，并导致了两者的鉴定规范和新的候选Cad11相互作用蛋白。p120连环蛋白与Cad11的缔合取决于所用的去污剂。使用等压定量相对和绝对定量（iTRAQ）来确定Cad11免疫复合物中蛋白质的相对水平，我们发现DDM和Triton X-100在Cad11的增溶和免疫沉淀中比胆酸盐更有效，并导致了两者的鉴定规范和新的候选Cad11相互作用蛋白。