Effects of the Mid peptide on the Doc2α–Munc13–1 interaction in a cell-free system. Affinity-purified glutathione S-transferase–Doc2α (amino acid residues 1–90) immobilized on glutathione–Sepharose beads was incubated with in vitro translated, [35S]-methionine-labeled Munc13–1 in the presence of the indicated concentrations of the Mid peptide or the control peptides. Specifically bound proteins were detected by SDS/PAGE followed by autoradiography. Mid, Mid peptide; m-Mid, mutated Mid peptide; s-Mid, scrambled Mid peptide. The protein markers used were myosin (Mr 200,000), β-galactosidase (Mr 116,000), and BSA (Mr 66,200). Results shown are representative of three independent experiments.