Protein-protein interactions play important roles in regulating cellular functions. We present an optimized disulfide cross-linking protocol for testing predicted interactions of soluble or membrane proteins. Coexpression in E. coli of proteins with a single cysteine residue results in disulfide bond formation upon treating the cells with oxidants if the two proteins interact and the cysteine residues are near each other. Quantification of cross-linked proteins after immunoblot sensitively and reproducibly measures the interaction. For complete details on the use and execution of this protocol, please refer to Olenic et al. (2022).1.
Keywords: Molecular Biology; Protein Biochemistry; Protein expression and purification.
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