TITLE: HisPur Purification of His-Tagged Proteins--CHEM 584
AUTHORS: Ken   Christensen

[DESCRIPTION]
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.

[GUIDELINES]
Important Product Information

 • Protein yield and purity are dependent upon the expression level, conformation and solubility characteristics of the recombinant fusion protein. Therefore, it is important to optimize these parameters before attempting a large-scale purification. For best results, perform a small-scale test to estimate the expression level and determine the solubility of each His-tagged protein. 

• Optimization of the lysis procedure is critical for maximizing protein yield. Some methods for protein extraction include using B-PER Bacterial Protein Extraction Reagent, mechanical methods such as freeze/thaw cycles, sonication or French press. 

• Sometimes overexpressed proteins are sequestered in inclusion bodies. Inclusion bodies of His-tagged proteins can be solubilized in 8M urea, 6M guanidine) and purified with the Ni-NTA resin, but a denaturant must be added to buffers so the protein remains soluble throughout the procedure. 

• These instructions are effective for many types of samples; however, optimization might be required to further reduce nonspecific binding. To optimize conditions, adjust the recommended imidazole concentration in the Equilibration, Wash and Elution Buffer. 

• Avoid using protease inhibitors or other additives that contain chelators, such as EDTA, or strong reducing agents, such as DTT or β-mercaptoethanol, which will disrupt the function of the nickel resin. 

• When using the Bradford Assay to monitor protein concentration in the elution fractions, dilute the samples at least 1:2 before performing the protein assay.

[STEPS]
1. Add 250 µL of a 50% Ni-NTA resinslurry to a 1.7 ml microcentrifuge tube. Centrifuge tube for 2 min at 700 × g and carefully remove and discard the supernatant.
 



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

3. Centrifuge tube for 2 min at 700 × g and carefully remove and discard buffer.

4. 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.
5. Add the prepared protein extract to the tube and mix on an end-over-end rotator for 30 minutes.
6. Centrifuge the tube for 2 min at 700 × g. If desired, save supernatant for downstream analysis.

7. Wash the resin with two resin-bed volumes of Wash Buffer. Centrifuge the tube for 2 min at 700 × g. Save supernatant for downstream analysis.

8. 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.
9. Elute bound His-tagged proteins using one resin-bed volume of Elution Buffer. Centrifuge tube for 2 min 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.