HisPur Purification of His-Tagged Proteins--CHEM 584

Abstract

The Thermo Scientific HisPur Ni-NTA Resin enables effective immobilized metal affinity chromatography (IMAC) purification of polyhistidine-tagged proteins from a soluble protein extract. This resin is composed of nickel-charged nitrilotriacetic acid (NTA) chelate immobilized onto 6% crosslinked agarose. The Ni-NTA resin is compatible with native or denaturing conditions and can be used in multiple formats, including conventional gravity-flow chromatography, spin column and FPLC. Ni-NTA resins are commonly chosen for His-tagged-protein purification because of the four metal-binding sites on the chelate, which allow for high-binding capacity and low-metal ion leaching.

Steps

Add 250µL of a 50% Ni-NTA resinslurry to a 1.7 ml microcentrifuge tube. Centrifuge tube for 0h 2m 0s at 700 × g and carefully remove and discard the supernatant.

Note

The HisPur Ni-NTA Resin allows for purification strategy customization. Purification conditions can be scaled as needed. The procedure may be performed at room temperature or at 4°C.It is important to spin the resin only at speeds up to 700 x gYou should only use a 1 ml pipette tip when manipulating the resin.

Add two resin-bed volumes of Equilibration Buffer and mix gently until the resin is fully resuspended

Note

Since you added 250µL of the 50% resin slurry to your tube, your resin bed volume is 125µL

Centrifuge tube for 0h 2m 0s at 700 × g and carefully remove and discard buffer.

Prepare sample by mixing the protein extract from your B-PER Lysis with an equal volume of Equilibration Buffer. The total volume should equal at least two volumes of the resin bed.

Add the prepared protein extract to the tube and mix on an end-over-end rotator for 30 minutes.

Centrifuge the tube for 0h 2m 0s at 700 × g. If desired, save supernatant for downstream analysis.

Wash the resin with two resin-bed volumes of Wash Buffer. Centrifuge the tube for 0h 2m 0s at 700 × g. Save supernatant for downstream analysis.

Repeat wash step. Optional: You can monitor the washing by measuring the absorbance of the supernatant by at 280 nm until a baseline is reached.

Elute bound His-tagged proteins using one resin-bed volume of Elution Buffer. Centrifuge tube for 0h 2m 0s at 700 × g. Carefully remove and save the supernatant. Repeat this step twice, saving each supernatant fraction in a separate tube.

10.

Monitor protein elution by measuring the absorbance of the fractions at 280nm. Since you are purifying a fluorescent protein, you can also monitor the protein concentration by measuring the absorbance at 500 nm.

Note

The eluted protein can be directly analyzed by SDS-PAGE. Samples containing 6M guanidine•HCl must be dialyzed against a buffer containing 8M urea before SDS-PAGE analysis.

Abstract

Steps

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