Kta explorer 100 fplc system

Yeast microsomes containing Strep-tagged DcUGT2 were isolated from a 250 ml culture, resuspended in 30 ml of binding buffer (100 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 1 mM EDTA), and solubilized by adding reduced Triton X-100 to a final concentration of 1% (v v⁻¹) under gentle stirring (1.5 h, 4 °C). The supernatant was isolated by ultracentrifugation (1 h, 105,000×g, 4 °C) and the Strep-tagged DcUGT2 affinity purified on an equilibrated 5 ml Strep-Tactin column (IBA GmBH), operated by an ÄKTA explorer 100 FPLC system (GE Life Sciences) and a flow rate of 1 ml min⁻¹. Column equilibration and washing were according to the manufacturer’s guidelines. Protein elution was carried out with a flow rate of 3 ml min⁻¹ using 10 column volumes of binding buffer containing 2.5 mM desthiobiotin in a gradient of 0–100%. Fractions (0.5 ml) were collected, desalted, analyzed by SDS-PAGE, and monitored for glucosylation activity using the described [¹⁴C]glucosylation enzyme assay.

Purified OsUAM2 was centrifuged and injected onto a Superdex 200 10/300 GL size-exclusion column (GE Life Sciences, catalog No. 17517501) using an ÄKTAexplorer 100 FPLC system (GE Life Sciences, catalog No. 18111241) and a flow rate of 0.5 ml min⁻¹. The column was pre-equilibrated in running buffer (25 mM HEPES, 300 mM NaCl, 5 mM MgCl₂ pH 8.0 with or without 1 mM DTT). Protein standards (Bio-Rad, catalog No. 151-1901) were run in the same buffers and with the same flow rate to ensure accurate results.