Rabdosianone I-Binding Protein Purification

Purification and identification of rabdosianone I-binding proteins were performed as previously described [24 (link)]. Briefly, HT-29 cells were lysed with binding buffer containing 50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1 mM DTT, and 0.43 mM ABSF at 4 °C for 30 min, and centrifuged. The supernatants were used as whole-cell extracts of HT-29 cells. The extracts were incubated with the agent-fixed beads or empty beads at 4 °C for 4 h. The beads were washed 3 times with binding buffer. The bound proteins were eluted with Laemmli dye and subjected to SDS-PAGE. The proteins were stained by aqueous AgNO₃, and each strip, including the protein, was cut off to apply Sequencing Grade Modified Trypsin (Promega, Madison, WI, USA). The peptide fragments from each strip were analyzed using an Autoflex II mass spectrometer (Bruker Daltonics, Billerica, MA, USA) after in-gel digestion.