Clarified and filtered culture media were applied to HisTrap Excel 5 ml column (Cytiva) at 3 ml/min followed by 10 CV wash with 50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, pH 8.0. Bound proteins were eluted with 50 mM sodium phosphate, 300 mM NaCl, 500 mM imidazole, pH 8.0. Pooled eluate was loaded onto Strep-Tactin Superflow Plus cartridge (1 ml or 5 ml at 0.5 ml/min or 3 ml/min; Qiagen). The resin was washed with 10 CV of 50 mM sodium phosphate, 300 mM NaCl, pH 8.0 and proteins were eluted in the same buffer with 2.5 mM desthiobiotin. Fractions containing protein were pooled, concentrated in 30 kDa MWCO concentrator (Pierce) to 0.1-0.4 ml and manually injected onto Superose 6 Increase (Cytiva) equilibrated in 20 mM Hepes, 150 mM NaCl, pH 7.6. Protein concentration was determined using Nanodrop 2000 or 8000 (Thermo Fisher).
Histrap excel 5 ml column
Manufactured by Cytiva
The HisTrap Excel 5 ml column is a prepacked affinity chromatography column designed for the purification of histidine-tagged recombinant proteins. The column contains Ni Sepharose High Performance resin, which has a high binding capacity and is suitable for both small-scale and large-scale purification.
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2 protocols using histrap excel 5 ml column
1
Purification of His-tagged Proteins
2
Anti-CD47 Fab Production and Purification
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Variable regions of heavy and light chains from anti-CD47 Fab P01A11_75 were sub-cloned into mammalian expression vectors encoding for (1) Fab CH1 domain followed by 8xHis-tag and (2) Fab constant lambda domain. Resulting vectors encoding His-tagged Fab fragment were transfected into HEK Expi293 cells and expressed for 5 days using standard expression protocols. Culture media from expression was harvested, and Fab purified on HisTrap excel 5 mL column (Cytiva), followed by size exclusion chromatography to remove any aggregates and to buffer exchange the Fab into PBS.
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