Analysis of Disulfide Bond Formation.
Ineke Braakman1, Lydia Lamriben2, Guus van Zadelhoff1
1Cellular Protein Chemistry, Bijvoet Center for Biomolecular Research, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
Current Protocols in Protein Science
|November 2, 2017
Summary
This study details methods for detecting disulfide bond formation in cells and in vitro systems. Protocols involve radioactive labeling, immunoprecipitation, and SDS-PAGE analysis to identify disulfide bonds in proteins.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Disulfide bonds are crucial for protein folding and stability.
- Accurate detection of disulfide bond formation is essential for understanding protein function and cellular processes.
Purpose of the Study:
- To provide detailed protocols for detecting disulfide bond formation.
- To enable analysis in both intact cell cultures and in vitro translation systems.
Main Methods:
- Biosynthetic labeling of newly synthesized proteins with radioactive amino acids.
- Isolation of proteins via immunoprecipitation after cell lysis.
- Analysis of disulfide bonds using SDS-PAGE with and without reduction.
- Utilizing PEG-maleimide to track disulfide bond formation via thiol modification.
Main Results:
- Demonstrated a difference in protein mobility on SDS-PAGE gels between reduced and nonreduced samples, indicating disulfide bond presence.
- Established protocols applicable to intact cells, isolated microsomes, and semi-permeabilized cells.
Conclusions:
- The provided protocols offer a robust method for assessing disulfide bond formation.
- These techniques facilitate the study of protein folding and stability in various biological contexts.
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