Superdex 75 hr 10 30 column

Multi-angle light scattering was used to probe for oligomerization states. All measurements were performed at room temperature using a Dawn Heleos multi-angle light scattering detector (Wyatt Technologies) coupled to an SEC column. 80 μL (0.5 mg mL⁻¹) of purified recombinant protein was injected into a Superdex 75 HR10/30 column (GE Healthcare) in buffer containing 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM TCEP, and eluted at a flow rate of 0.5 mL min⁻¹. Absolute molecular weight and homogeneity were determined using ASTRA version 5.3 (Wyatt Technologies).

SEC was performed with a Superdex 75HR 10/30 column (GE Healthcare). Purified His-FlhA_C and its mutant variants (10 μM) were run on the SEC column equilibrated with 50 mM Tri-HCl, pH 8.0, 150 mM NaCl at a flow rate of 0.5 ml min⁻¹. γ-Globulin (158 kDa), bovine serum albumin (66.4 kDa) and ovalbumin (43 kDa) were used as size markers. All fractions were run on SDS-PAGE and then analyzed by Coomassie Brilliant blue (CBB) staining.

A water extract of gold beans Phaseolus vulgaris cv. “gold bean” from Mainland China (260 g) was made by homogenizing them in distilled water (6 mL/g). The homogenate was then centrifuged (14,000× g for 25 min at 4 °C). The supernatant was collected and loaded on a 5 × 20 cm column of DEAE-cellulose (Sigma, St. Louis, MI, USA) in 10 mM Tris-HCl buffer (pH 7.4). Following removal of unadsorbed proteins (fraction D1), the column was eluted sequentially with 0.2 M NaCl and 1 M NaCl in the Tris-HCl buffer. Fraction D2 eluted with 0.2 M NaCl was dialyzed and then chromatographed on a 5 × 15 cm column of Affi-gel blue gel (Bio-Rad, Woodinville, WA, USA) in 10 mM Tris-HCl buffer (pH 7.4). The unadsorbed proteins (fraction B1) were dialyzed against 10 mM NH₄Ac buffer (pH 5) and applied on a 2.5 × 20 cm column of SP-sepharose (GE Healthcare, Uppsala, Sweden). After elution of unadsorbed proteins (fraction S1), the column was eluted with a 0–1 M NaCl concentration gradient in the NH₄Ac buffer. The first adsorbed fraction (S2) was then subjected to gel filtration on a Superdex 75 HR 10/30 column (GE Healthcare) in 0.2 M NH₄HCO₃ buffer (pH 8.5). The second absorbance peak (SU2) represented purified trypsin inhibitor (GBTI).

The molecular weight of the Trx variants has been determined by size exclusion chromatography on a Superdex 75 HR 10/30 column connected to an ÄTKA explorer (GE healthcare, Bio-Sciences, Pittsburgh, USA). 100 μl of the Ni-NTA elution fractions were loaded and eluted in gel filtration buffer (20 mM HEPES–NaOH, pH 7.7, 150 mM NaCl) at a flow rate of 0.5 ml/min.