Purification and Cross-linking of Glt(Ph) Transporter

Glt_Ph-K55C/C321A/A364C was expressed as His₈ fusion and purified as described previously8 (link). Transporter samples were exchanged by size exclusion chromatography (SEC) into buffer, containing (in mM) 10 HEPES/NaOH or KOH, pH 7.4, 1 n-dodecyl-β-D-maltopyranoside and either 100 NaCl and 0.1 L-asp or 100 KCl. Cross-linking was initiated by addition of 1:2 molar ratio of Cu²⁺ and 1,10 phenantroline or HgCl₂. Reactions were quenched with 100 mM N-ethyl maleimide prior to SDS PAGE analysis. Crude E. coli membranes were isolated by centrifugation, washed either in a Na⁺/L-asp-containing or free buffer and cross-linked as in detergent. Protein bands were visualized by western blotting using antibodies against histidine tag.