Figure 4

Analysis of self-associating properties of BnDGAT1_(1–116)His₆ using gel filtration chromatography and chemical cross-linking. Panel A: Gel filtration chromatography of clarified lysate of bacteria overexpressing BnDGAT1_(1–116)His₆ E. coli producing BnDGAT1_(1–116)His₆ and SDS-PAGE of protein in fractions eluted from the column. IPTG-induced bacteria were resuspended in 30 ml of 100 mM sodium phosphate buffer (pH 7.4) containing 150 mM NaCl. The suspension was passed through a French press three times at 20,000 p.s.i.. The suspension of ruptured bacteria was centrifuged at 13,000 g for 15 min to obtain a clarified lysate. The Superdex 75 HR 10/30 column was equilibrated with extraction buffer (100 mM sodium phosphate, pH 7.4 containing 150 mM NaCl) and operated at room temperature with a flow rate of 0.5 mL/min using an FPLC system. One hundred microliters of extract were applied to the column and 1 ml fractions were collected during elution. Ten microliter aliquots were analyzed by SDS-PAGE using a separating gel prepared with a concentration of 15% monomer and 1.1% cross-linker. The column and SDS gel were calibrated with molecular mass markers. Panel B: Chemical cross-linking of BnDGAT1_(1–116)His₆ monitored by Western blotting of BnDGAT1_(1–116)His₆ before (lane 1) and after (lane 2) treatment with DMS. BnDGAT1_(1–116)His₆ (500 μg/mL) was cross-linked in the presence of 4 mg DMS per ml in 0.2 M triethanolamine-HCl at pH 8.5. Cross-linking reactions were allowed to proceed at room temperature for 3 h. The reactions were quenched with 2 × SDS loading buffer and boiled for 5 min prior to application of 10 μL samples to an SDS-PAGE gel, which was prepared using a concentration of 10% total monomer and 1.1% cross-linker.