1. Protein Purification by Nickel Affinity Chromatography
<ol>
	<li>Purify protein using 1 mL of Nickel-Nitriloacetic Acid (Ni-NTA) resin on a gravity column.
	<ol>
		<li>The day before use, equilibrate the column overnight at 4 &#176;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).</li>
		<li>The following day bring the column from 4 &#176;C to RT prior to loading the clarified lysate and let it stand for ~2&#8211;3 h. 
		NOTE: Bringing the column to thermal equilibrium is crucial to avoid air bubbles forming within the column.</li>
		<li>Resuspend the pellet in the lysis buffer (10 mM Tris-HCl pH 7.8, 300 mM NaCl, 5 mM MgCl2, 50 mM NaH2PO4, 10% glycerol, 0.5 mg/mL lysozyme, 10 mM imidazole, 0.25 mM DTT and 5 mM PMSF).</li>
		<li>Sonicate cells on ice for 8 x 10 s intervals, pausing 30 s between pulses.</li>
		<li>Clarify the lysate by centrifugation at 3,080 x g for 30 min at 4 &#176;C using a microcentrifuge.</li>
		<li>Prepare lysate with equal volumes of lysis buffer then apply the prepped clarified lysate to the column and collect the flow-through.</li>
		<li>Reapply clarified lysate flow-through to the column and collect the secondary flow-through.</li>
		<li>Wash the column with wash buffer 1 (10 mM Tris-HCl (pH 7.79), 300 mM NaCl, 5 mM MgCl2, 50 mM NaH2PO4, 30 mM imidazole, 10% glycerol). Collect the flow-through. 
		NOTE: Inclusion of 5 mM MgCl2 in the wash and elution buffers is important for enzymatic activity of the purified protein.</li>
		<li>Wash the column with wash buffer 2 (10 mM Tris-HCl (pH 7.79), 300 mM NaCl, 5 mM MgCl2, 50 mM NaH2PO4 and 50 mM imidazole).</li>
		<li>Collect the flow-through.</li>
		<li>Apply 2 mL elution buffer (10 mM Tris-HCl (pH 7.79), 300 mM NaCl, 50 mM NaH2PO4, 10% glycerol and 75 mM imidazole). Collect flow-through in two fractions of 1 mL each. 
		NOTE: The column after this step can be stored in equilibration buffer at 4 &#176;C if the same protein will be purified in the next purification assay. The column can be used up to 3 times if stored properly.</li>
	</ol>
	</li>
</ol>

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

nickel affinity chromatography-based protein purification: a technique to purify polyhistidine-tagged recombinant proteins from bacterial cell lysate

Source: Pokhrel, A. et al. A <a href='https://www.jove.com/v/58547'>Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile</a>. J. Vis. Exp. (2018)
In this video, we demonstrate the nickel affinity chromatography technique to purify histidine-tagged pyrophosphokinase enzymes from Clostridium difficile bacteria.

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

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 ...

To purify recombinant protein fused to poly-histidine tag - a string of histidine residues at one terminus - from a bacterial cell lysate, first, dilute the cell lysate to prevent column clogging during chromatographic run.
Assemble an affinity column containing divalent nickel cations immobilized onto an agarose bead matrix using nitrilotriacetic acid, a chelating agent. Nitrilotriacetic acid binds nickel cations via four coordinate covalent sites, while two sites on the metal ion remain available for interactions with poly-histidine tags in the protein of interest.
Equilibrate the column with suitable buffer to optimize conditions for effective metal-protein interactions. Load the cell lysate onto the column.
Recombinant proteins, carrying accessible terminal histidine residues, form coordination bonds with nickel cations due to the metal ion's high affinity for imidazole, the side-chain of histidine, and bind to the matrix. The high number of consecutive histidines in the recombinant protein strengthens its attachment to the matrix.
In comparison, proteins lacking histidine residues do not adhere to the column and elute in flow-through. Wash the column with low-concentrated imidazole buffer.
Imidazole competes with weakly bound protein contaminants that may have nonspecifically adhered to the matrix via histidine residues in their polypeptide chain, displacing them and facilitating elution. Wash the column with highly concentrated imidazole buffer to dissociate the strongly-bound histidine-tagged recombinant proteins from nickel ion chelate, eluting them in flow-through.&#160;
Collect the purified recombinant protein fraction for further analysis.

Begin this protocol with inducible over-expression of a histidine-tagged protein as described in the text protocol. Prepare 1 milliliter of Nickel-Nitriloacetic Acid resin in a gravity column, to purify the protein. The day before use, equilibrate the column overnight at 4 degrees Celsius with 2 milliliters of equilibration buffer.
The following day, bring the column from 4 degrees Celsius to room temperature, prior to loading the clarified lysate, and let it stand for approximately two to three hours. Then, re-suspend the pellet in the lysis buffer.
Sonicate the cells on ice for 10 times 10-second intervals, pausing 30 seconds between pulses. Clarify the lysate by centrifugation at 3080 x g for 30 minutes at 4 degrees Celsius using a microcentrifuge.
Prepare the clarified lysate with equal volumes of lysis buffer, then, apply the prepped clarified lysate to the column, and collect the flow-through. Re-apply the clarified lysate flow-through to the column, and collect the secondary flow-through.
Then, wash the column with 5 milliliters of wash buffer #1 and collect the flow-through. Wash the column again with 5 milliliters of wash buffer #2, and collect the flow-through. Now, apply 2 milliliters of elution buffer. Collect flow-through in two fractions of 1 milliliter each.

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7.4K Views. To purify recombinant protein fused to poly-histidine tag - a string of histidine residues at one terminus - from a bacterial cell lysate, first, dilute the cell lysate to prevent column clogging during chromatographic run. Assemble an affinity column containing divalent nickel cations immobilized onto an agarose bead matrix using nitrilotriacetic acid, a chelating agent. Nitrilotriacetic acid binds nickel cations via four coordinate covalent sites, while two sites on the metal ion rem...

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

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Name	Company	Catalog Number
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