Nickel Affinity Chromatography-Based Protein Purification: A Technique to Purify Polyhistidine-Tagged Recombinant Proteins from Bacterial Cell Lysate
Published: April 30, 2023
Abstract
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 Purify protein using 1 mL of Nickel-Nitriloacetic Acid (Ni-NTA) resin on a gravity column. 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). The following day bring the column…
Divulgaciones
The authors have nothing to disclose.
Materials
| 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 |
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Nickel Affinity Chromatography-Based Protein Purification: A Technique to Purify Polyhistidine-Tagged Recombinant Proteins from Bacterial Cell Lysate. J. Vis. Exp. (Pending Publication), e21093, doi: (2023).