TY - JOUR
T1 - Impact of Detergents on Membrane Protein Complex Isolation
AU - Lee, Yu Chen
AU - Bååth, Jenny Arnling
AU - Bastle, Ryan M.
AU - Bhattacharjee, Sonali
AU - Cantoria, Mary Jo
AU - Dornan, Mark
AU - Gamero-Estevez, Enrique
AU - Ford, Lenzie
AU - Halova, Lenka
AU - Kernan, Jennifer
AU - Kürten, Charlotte
AU - Li, Siran
AU - Martinez, Jerahme
AU - Sachan, Nalani
AU - Sarr, Medoune
AU - Shan, Xiwei
AU - Subramanian, Nandhitha
AU - Rivera, Keith
AU - Pappin, Darryl
AU - Lin, Sue Hwa
N1 - Publisher Copyright:
© 2017 American Chemical Society.
PY - 2018/1/5
Y1 - 2018/1/5
N2 - 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 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.
AB - 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 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.
KW - LC-MS/MS
KW - cadherin-11
KW - detergents
KW - iTRAQ
KW - membrane protein complex
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U2 - 10.1021/acs.jproteome.7b00599
DO - 10.1021/acs.jproteome.7b00599
M3 - Article
C2 - 29110486
AN - SCOPUS:85040173548
SN - 1535-3893
VL - 17
SP - 348
EP - 358
JO - Journal of Proteome Research
JF - Journal of Proteome Research
IS - 1
ER -