Lee, Y. C., Baath, J. A., Bastle, R. M., Bhattacharjee, S., Cantoria, M. J., Dornan, M., Gamero-Estevez, E., Ford, L., Halova, L., Kernan, J., Kurten, C., Li, S., Martinez, J., Sachan, N., Sarr, M., Shan, X., Subramanian, N., Rivera, K., Pappin, D., Lin, S. H. (January 2018) Impact of Detergents on Membrane Protein Complex Isolation. J Proteome Res, 17 (1). pp. 348-358. ISSN 1535-3893
Abstract
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. Furthermore, we compared the effects of Brij-35, Triton X-100, cholate, CHAPSO, Zwittergent 3-12, Deoxy BIG CHAP, and digitonin on Cad11 solubilization and immunoprecipitation. We found that all detergents except Brij-35 could solubilize Cad11 from the membrane. Upon immunoprecipitation, we found that beta-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.
| Item Type: | Paper |
|---|---|
| Uncontrolled Keywords: | Lc-ms/ms cadherin-11 detergents iTRAQ membrane protein complex |
| Subjects: | bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification Investigative techniques and equipment > spectroscopy > mass spectrometry bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein characterization |
| CSHL Authors: | |
| Communities: | CSHL labs > Martienssen lab CSHL labs > Pappin lab |
| Depositing User: | Matt Covey |
| Date: | 5 January 2018 |
| Date Deposited: | 26 Jan 2018 20:00 |
| Last Modified: | 08 Oct 2020 19:31 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/35974 |
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