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Impact of Detergents on Membrane Protein Complex Isolation
Yu-Chen Lee1, Jenny Arnling Bååth2, Ryan M Bastle2
1Department of Translational Molecular Pathology, The University of Texas M. D. Anderson Cancer Center , Houston, Texas 77030, United States.
Journal of Proteome Research
|November 8, 2017
Summary
Choosing the right detergent is crucial for isolating membrane protein complexes like cadherin-11 (Cad11). Different detergents significantly impact protein interactions and antibody binding during immunoprecipitation, affecting downstream analysis.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Detergents are essential for solubilizing membrane proteins for analysis.
- The choice of detergent can influence protein structure, antibody binding, and protein-protein interactions.
- Cadherin-11 (Cad11) serves as a model to investigate detergent effects on membrane protein complex isolation.
Purpose of the Study:
- To evaluate the impact of various detergents on the solubilization and immunoprecipitation of Cadherin-11 (Cad11).
- To assess how different detergents affect the interaction of Cad11 with its binding partners, such as beta-catenin and p120 catenin.
- To identify optimal detergents for Cad11 complex isolation and proteomic analysis.
Main Methods:
- Solubilization of membrane protein complexes using a panel of detergents including dodecyl maltoside (DDM), octylglucoside, Brij-35, Triton X-100, cholate, CHAPSO, Zwittergent 3-12, Deoxy BIG CHAP, and digitonin.
- Immunoprecipitation of Cadherin-11 (Cad11) using a specific monoclonal antibody (mAb 1A5).
- Proteomic analysis of co-immunoprecipitated proteins using isobaric tag for relative and absolute quantitation (iTRAQ).
Main Results:
- Dodecyl maltoside (DDM) and Triton X-100 demonstrated superior efficiency in Cad11 solubilization and immunoprecipitation compared to cholate.
- Octylglucoside interfered with the Cad11-mAb 1A5 interaction, preventing immunoprecipitation.
- Beta-catenin consistently co-immunoprecipitated with Cad11 across most detergents, while p120 catenin association varied.
- iTRAQ analysis identified known and novel Cad11-interacting proteins, with DDM and Triton X-100 yielding richer datasets.
Conclusions:
- Detergent selection critically influences the success of membrane protein complex isolation and subsequent proteomic studies.
- Dodecyl maltoside (DDM) and Triton X-100 are effective detergents for Cadherin-11 (Cad11) complex isolation and interaction profiling.
- Understanding detergent-specific effects is vital for accurate characterization of membrane protein interactions and functions.
