Nickel Affinity Chromatography-Based Protein Purification: A Technique to Purify Polyhistidine-Tagged Recombinant Proteins from Bacterial Cell Lysate

Samenvatting

Source: Pokhrel, A. et al. A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile. J. Vis. Exp. (2018)

In this video, we demonstrate the nickel affinity chromatography technique to purify histidine-tagged pyrophosphokinase enzymes from Clostridium difficile bacteria.

Protocol

1. Protein Purification by Nickel Affinity Chromatography

1. Purify protein using 1 mL of Nickel-Nitriloacetic Acid (Ni-NTA) resin on a gravity column.
   1. The day before use, equilibrate the column overnight at 4 °C with 2 mL of equilibration buffer (10 mM Tris-HCl pH 7.79, 300 mM NaCl, 50 mM NaH₂PO₄, 0.5 mg/mL lysozyme, 5 mM MgCl₂, 10 mM imidazole, 0.25 mM DTT, 5 mM phenylmethane sulfonyl fluoride (PMSF), 10% glycerol).
   2. The following day bring the column from 4 °C to RT prior to loading the clarified lysate and let it stand for ~2–3 h.
      NOTE: Bringing the column to t....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Ni-NTA resin	G Biosciences	786-940/941
Pierce Disposable Gravity columns, 10 mL	Thermo Scientific	29924
1 mL Spectra/ Por float-A-lyzer G2 dialysis device (MWCO: 20-kD)	Spectrum	G235033
Ultrasonic processor	Sonics	VC-750
Microcentrifuge with D3024/D3024R rotor	Scilogex