Protein assays kits 5000001

The recombinant mBPI5 and its mutant were expressed in E. coli strain SHuffle. Cells were grown at 37°C in LB medium containing 50 μg/ml Ampicillin to an OD600 of 0.6, and were then induced by the addition of 0.1 mM isopropyl β-D- thiogalactoside (IPTG) at 16°C for 12 h. Cells were harvested and re-suspended in buffer (25 mMTris-HCl pH 8.0, 300 mM NaCl), disrupted by sonication and centrifuged at 12000 g for 20 min at 4°C. Recombinant protein was exclusively detected in the precipitates. BPIs inclusion bodies were resuspended in 20 ml solubilization buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, 8 M urea, pH 8.0), incubated by stirring for 2 h at room temperature, and then centrifuged at 12000 g for 20 min. The supernatant was applied to a nickel affinity column(Qiagen WorkBeads^™ 40 Ni #40650003) equilibrated with the same buffer. Removal of contaminating proteins and initial refolding were carried out on the column by exchanging to nondenaturing buffer conditions with a linear 8–0 M urea gradient before elution of the bound protein. The recombinant BPIs was eluted with elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, 300 mM imidazole, pH 8.0) and concentrated by ultrafiltration (Amicon #UFC500308), The eluted products were determined by Bradford protein assays (Bio-Rad Protein Assays Kits #5000001) analyzed by 15% reduced and non-reduced SDS-PAGE.

Liposome co-sedimentation assay was performed as described previously [21 (link)]. Lipid mixes (Avanti Polar Lipids) were suspended at 1 mg/ml in buffer containing 25 mM Hepes pH 7.4, 100 mM NaCl and 1 mM dithiothreitol, and liposomes were formed by sonication followed by hydration. Purified proteins were incubated with liposomes for 15 min at room temperature and were centrifuged at 60000 rpm for 20 min at 25°C. Obtained supernatants and pellets were subject to SDS-PAGE. The ratio of the amount of protein binding to lipid to the amount of protein in supernatants was determined using the Bradford protein assays (Bio-Rad Protein Assays Kits #5000001).