In Vitro Cleavage Assay Using g-CPD Biosensor to Detect InsP6 v1

<p>This protocol describes an in vitro autocatalytic cleavage assay to evaluate endogenous inositol hexakisphosphate (InsP6) bound to DNAJC13 protein. The Clostridium difficile Toxin A glucosyltransferase-cysteine protease domain (g-CPD) is used as a molecular biosensor. Recombinant DNAJC13(1-1486)- WT and InsP6-binding mutant DNAJC13(1-1486)-8A are incubated with g-CPD under defined conditions, and InsP6-dependent autoprocessing of g-CPD is monitored over time by SDS-PAGE and western blotting.</p>