Figure 2.

Identification of α-SNAP cross-links. (A) Complete incubations containing carbonate-washed brain membranes were recovered on protein G-agarose and analyzed by SDS PAGE (control). Alternatively, samples were subjected to secondary immunoprecipitations using monoclonal anti-NSF or anti-SNAP25 antibodies. For secondary precipitations, twice the amount of sample was used, to compensate for loss of material during the second precipitation step. (B) Samples containing membranes, His₆-NSF-myc, and unmodified [³⁵S]-α-SNAP were incubated with SMCC. After quenching with glycine, 20S complexes were recovered and analyzed by SDS-PAGE (lane 1). Alternatively, a secondary immunoprecipitation step was performed with anti-syntaxin antibody before analysis (lane 2).