Superose 12 10 300 gl column

Purified recombinant Pdr16 proteins were subjected to size exclusion chromatography on a Superose™12 10/300 GL column (GE Healthcare) equilibrated with 20 mM PIPES pH 6.8, 250 mM NaCl, 2.7 mM KCl buffer. Proteins were monitored at 280 nm. The flow rate was 0.4 ml/min.

Size exclusion chromatography was performed using AktaPure and a Superose 12 10/300 GL column (GE Healthcare, Inc). A buffer consisting of 10 mM HEPES pH 8.0, 100 mM NaCl, and 10 mM MgCl₂ was used to run ≈10–20 μM (0.2–0.5 mg/mL) protein over the column at 0.1–0.25 mL/min. Additional 1 mM DTT was used for proteins containing cysteines. For gel filtration with ligands, 500 μM PRPP and 100 μM 9-deazaguanine were included in the mobile phase buffer where necessary. A gel filtration standard (Bio-Rad) was used to establish molecular weight, and bovine serum albumin (BSA) was included as an additional marker.

Size-exclusion chromatography assays were performed with an AKTA purifier system by using a Superose^® 12 10/300 GL column (GE Healthcare) equilibrated with 50 mM Tris–HCl pH 8.0 and 150 mM NaCl. The flow rate for protein elution was 1 mL/min. β-Amylase (200 kDa), β,γ-globulin (158 kDa), bovine serum albumin (66 kDa) and carbonic anhydrase (29 kDa) were used as molecular mass standards. Apparent Mr values of enzymes were obtained from a graph where the elution volume (Ve) of the standard proteins was plotted against log MW.

The HtrA variants (10 μM monomer) were preincubated for 15 minutes at 37°C with or without a 22-peptide ligand in 20 mM HEPES-NaOH pH 8.0, 100 mM NaCl, and then incubated for 10 hours at 4°C. Then, the samples were treated with the addition of BS³ to a final concentration of 0.45 mM and incubated at room temperature for 30 minutes. The cross-linking reaction was terminated by the addition of Tris-HCl buffer (pH 7.5) to a final concentration of 50 mM.
SEC was carried out on a Superose 12 10/300 GL column (GE Healthcare) equilibrated with 50 mM Tris-HCl, pH 8.0, 100 mM NaCl. 50 μl samples of the cross-linked HtrA species were analyzed at 25°C at a flow rate of 0.3 ml/min.
The elution volumes of molecular weight standards (Bio-Rad) were used for column calibration and calculation of the HtrA monomer level in each eluted cross-linked species.

10 μM Clamp module were mixed with 10 μM of SsoTFEαβ or mutants thereof in TK(250) buffer, 10% glycerol, 10 mM MgCl₂ and incubated at 37°C for 10 min. After brief centrifugation, 250 μl were loaded on a Superose 12 10/300 GL column (GE Life Sciences) equilibrated in the same buffer plus 0.05% TWEEN 20. The fraction size was 0.5 ml. The elution profile of the respective proteins was visualized on silver-stained SDS-PAGE gels.

Analytical gel filtration chromatography was carried out on an AKTA FPLC system (GE Healthcare). Protein samples were loaded on to a Superose 12 10/300 GL column (GE Healthcare) equilibrated with a buffer containing 20 mM Tris-HCl (pH 7.5), 100 mM NaCl and 1 mM dithiothreitol (DTT).

For MALS measurement, OPTN(1–119), OPTN(1–119) E50K mutant, Trx-tagged TBK1(677–729) or Trx-tagged TBK1(677–729) E696K mutant sample (100 μl at a concentration of 20 μM) was injected into an AKTA FPLC system with a Superose 12 10/300 GL column (GE Healthcare) with the column buffer containing 50 mM Tris-HCl, 100 mM NaCl, 1 mM DTT and 1 mM EDTA at pH 7.5. The chromatography system was coupled to a static light-scattering detector (miniDawn, Wyatt Technology) and a differential refractive index detector (Optilab, Wyatt Technology). Data were collected every 0.5 s with a flow rate of 0.5 ml min⁻¹. Data were analysed using ASTRA 6 (Wyatt Technology, USA).