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Native Elution of Yeast Protein Complexes Obtained by Affinity Capture
John LaCava1, Javier Fernandez-Martinez1, Michael P Rout1
1Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York 10065.
Cold Spring Harbor Protocols
|July 3, 2016
Summary
This protocol details two methods for native protein complex elution after affinity capture. These methods, using protease cleavage or competitive reagents, yield purer, though potentially less efficient, native protein assemblies.
Area of Science:
- Biochemistry
- Molecular Biology
- Proteomics
Background:
- Affinity capture is crucial for isolating protein complexes.
- Native elution preserves complex integrity for downstream analysis.
- Existing elution methods may compromise complex structure or purity.
Purpose of the Study:
- To describe two distinct protocols for the native elution of protein complexes.
- To provide methods that maintain the structural integrity of purified protein assemblies.
- To offer alternatives to denaturing elution methods.
Main Methods:
- Elution using human rhinovirus 3C protease (PreScission protease) cleavage of tagged protein complexes.
- Competitive elution of protein A-tagged complexes using PEGylOx reagent.
- Comparison of native elution efficiency and purity against denaturing methods.
Main Results:
- Both protease-mediated and competitive elution successfully released native protein complexes.
- Native elution resulted in higher purity of eluted complexes compared to denaturing methods.
- Elution efficiency was dependent on the specific complex, affinity capture method, and reagent used.
Conclusions:
- Native elution protocols using protease or competitive reagents are effective for purifying intact protein complexes.
- While potentially less efficient than denaturing methods, native elution enhances purity by reducing non-specific binding.
- Consideration of protease or elution peptide carryover is necessary for downstream applications.
