Superdex 200 10 300 gl 24 ml column

The oligomeric state of glutathionylated, disulfide-bonded, and reduced WT or mutant hHsp70 or hHsc70 were compared by SEC using a Superdex 200 10/300 Gl 24 ml column (GE) for full length Hsp70 proteins or a Superdex 75 10/300 Gl 24 ml column (GE) for truncated Hsp70 mutants, in buffer B at RT. Blue dextran (2000 kDa), beta-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), PMSF-treated trypsinogen (24 kDa), and cytochrome c (12.4 kDa) were used as molecular mass standards.

Transaminase pQR2189 was expressed on a 400 mL scale as previous described. After centrifugation cells were resuspended in 5 mL buffer (Na₂HPO₄ 50 mM, NaCl 500 mM, imidazole 10 mM, pH 7.4, 0.5 mM PLP) and lysed by sonication (4 × 45 s). Cell debris was removed via centrifugation (45 min, 10 500 rpm, 4 °C) and the cell lysate loaded into an open tubal mini column containing Chelating Sepharose™ Fast Flow resin, and the column washed with 50 mL of start buffer. Proteins were eluted with elution buffer (Na₂HPO₄ 50 mM, NaCl 500 mM, imidazole 50–500 mM, pH 7.4) with increasing concentrations of imidazole. The eluents with the highest concentrations of protein were pooled and dialysed overnight at 4 °C into dialysis buffer (Tris 20 mM, NaCl 150 mM, PLP 0.5 mM, pH 7.5). Protein was concentrated using a 10 kDa cut off Vivaspin concentrator (Sartorius, Germany), and purified further by size exclusion chromatography using a Superdex 200 10/300 GL 24 mL column (GE Healthcare). The fractions were pooled and concentrated to 24 mg mL^–1 and stored at –80 °C.