Nanodrop extinction co efficient method

For His6-tagged proteins, 500 mL cultures of E. coli containing the relevant expression plasmid were induced at mid-exponential growth phase with 0.2 mM IPTG overnight at 20°C (Fogg and Wilkinson, 2008 (link)). Concentrated cells were lysed in 20 mL binding buffer (0.5 M NaCl, 75 mM Tris; pH 7.75) plus 0.2 mg mL⁻¹ lysozyme and 500 U Basemuncher Endonuclease (Expedeon Ltd.) for 30 min on ice and then sonicated. Cleared supernatant was applied to a 5 mL HisTrap FF crude column (Cytiva) and the bound, his-tagged protein was eluted with 125 mM imidazole. Eluted protein was desalted on a HiPrep 26/10 desalting column (Cytiva) and then further separated by size exclusion chromatography on a HiLoad 16/60 Superdex 200 preparative grade gel filtration column. All chromatography steps were carried out on an AKTA Prime instrument (Cytiva). Purified proteins were concentrated in a Spin-X UF Centrifugal Concentrator (Corning) and quantified by the nanodrop extinction co-efficient method (Thermo Scientific). Samples were stored at −80°C in binding buffer plus 50% glycerol. MBP-tagged proteins were purified as above except MBP binding buffer was used (200 mM NaCl, 20 mM Tris, 1 mM EDTA; pH 7.4), the lysate was applied to a 5 mL MBPTrap FF column (Cytiva) and purified protein was eluted with 10 mM maltose in binding buffer.

For His6-tagged proteins, 500 ml cultures of E. coli containing the relevant expression plasmid were induced at mid-exponential growth phase with 0.2 mM IPTG overnight at 20 °C. Concentrated cells were lysed in 20 ml binding buffer (1 M NaCl, 75 mM Tris; pH 7.75) plus 0.2 mg ml⁻¹ lysozyme and 500 U Basemuncher Endonuclease (Expedeon Ltd.) for 30 min on ice and then sonicated. Cleared supernatant was applied to a 5 ml HisTrap FF crude column (GE Healthcare) and the bound, his-tagged protein was eluted with 125 mM imidazole. Eluted protein was desalted on a HiPrep 26/10 desalting column (GE Healthcare) and then further separated by size exclusion chromatography on a HiLoad 16/60 Superdex 200 preparative grade gel filtration column. All chromatography steps were carried out on an AKTA Prime instrument (GE Healthcare). Purified proteins were concentrated in a Spin-X UF Centrifugal Concentrator (Corning) and quantified by the nanodrop extinction co-efficient method (Thermo Scientific). Samples were stored at −80 °C in binding buffer plus 50% glycerol. MBP-tagged proteins were purified as above except the cells were induced with 1 mM IPTG, MBP binding buffer was used (200 mM NaCl, 20 mM Tris, 1 mM EDTA; pH 7.4), the lysate was applied to a 5 ml MBPTrap FF column (GE Healthcare) and purified protein was eluted with 10 mM maltose in MBP binding buffer.