Tris 2 carboxyethyl phosphine

A 200 μL sample containing 25 μM MBP-tagged BalhC/D was prepared in cleavage buffer [20 mM Tris (pH 7.5), 500 mM NaCl, 10 % glycerol (v/v), 0.5 mM tris(2-carboxyethyl)phosphine (Gold Biotechnology)] and treated with 0.05 mg/mL TEV protease for 12 h at 4 °C. The amount of protein in a BalhC/BalhD complex was assessed on a Flexar HPLC (Perkin Elmer) equipped with an analytical Yarra SEC-3000 (300 × 4.6 mm, Phenomenex) equilibrated with cleavage buffer. Peaks of interest were collected and their composition was determined via a Coomassie stained 12% SDS-PAGE gel. The approximate molecular weights were determined by generating a standard curve with a 12–200 kDa molecular weight standard kit (Sigma). Control runs were also performed in which one of the two proteins was omitted. Chromatograms were analyzed using Flexar Manager (Perkin Elmer).

Recombinant t-and v-SNARE proteins were expressed in Escherichia coli and purified by nickel affinity chromatography. The synaptic t-SNARE complex was composed of His₆-SNAP-25 and untagged syntaxin-1. The v-SNARE VAMP2 possessed no extra residues after the tag was proteolytically removed^{34 (link)}. SNAREs were stored in a buffer containing 25 mM HEPES (pH 7.4), 400 mM KCl, 1% n-octyl-β-d-glucoside (Calbiochem), 10% glycerol (v/v) and 0.5 mM Tris(2-carboxyethyl)phosphine (Gold Biotechnology). The cytoplasmic domains (CDs) of SNAREs were purified using the same procedures as the WT proteins except that detergents were not included. Recombinant Munc18-1 proteins were expressed and purified from E. coli. The bacteria were grown at 37 °C until the optical density reached 0.6. Subsequently the temperature was lowered to 16 °C and 0.2 mM isopropyl-β-d-thiogalactoside was added to induce Munc18-1 expression. After 16 h, the bacteria were harvested and Munc18-1 proteins were purified using affinity chromatography. SNARE and Munc18-1 mutants were generated by site-directed mutagenesis (Stratagene/Agilent Technologies), and purified similarly to the corresponding WT proteins.