Protocol for identification and validation of 2′3′-cGAMP-binding proteins by photoaffinity probes

Mammalian cyclic dinucleotide 2′3′-cGAMP functions as a second messenger in innate immune response. Here, we report a protocol to utilize 2′3′-cGAMP photoaffinity probes to capture 2′3′-cGAMP-binding or 2′3′-cGAMP-interacting proteins from HeLa cell lysate for in-gel visualization by fluorescent imaging or identification by SILAC–based quantitative MS. Further validation is also executed using photoaffinity probes to demonstrate the direct interaction of 2′3′-cGAMP with purified target proteins in vitro or endogenous target proteins in 293T cells.