Mar 07, 2022
HisPur Purification of His-Tagged Proteins--CHEM 584
- 1Brigham Young University
Protocol Citation: Ken Christensen 2022. HisPur Purification of His-Tagged Proteins--CHEM 584. protocols.io https://dx.doi.org/10.17504/protocols.io.b52mq8c6
Manuscript citation:
https://www.thermofisher.com/order/catalog/product/88221#/88221
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: March 07, 2022
Last Modified: March 07, 2022
Protocol Integer ID: 59181
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.
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.
Materials
For native conditions prepare the following buffers:
• Equilibration Buffer: 20mM sodium phosphate, 300mM sodium chloride (PBS) with 10mM imidazole; pH 7.4
• Wash Buffer: PBS with 25mM imidazole; pH 7.4
• Elution Buffer: PBS with 250mM imidazole; pH 7.4
For denaturing conditions prepare the following buffers:
• Equilibration Buffer: PBS with 6M guanidine•HCl and 10mM imidazole; pH 7.4
• Wash Buffer: PBS with 6M guanidine•HCl and 25mM imidazole; pH 7.4
• Elution Buffer: PBS with 6M guanidine•HCl and 250mM imidazole; pH 7.4
For resin regeneration prepare the following buffer:
• MES Buffer: 20mM 2-(N-morpholine)-ethanesulfonic acid, 0.1M sodium chloride; pH 5.0
Add 250 µL of a 50% Ni-NTA resinslurry to a 1.7 ml microcentrifuge tube. Centrifuge tube for 00:02:00 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 g
You 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 250 µL of the 50% resin slurry to your tube, your resin bed volume is 125 µL
Centrifuge tube for 00:02:00 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 00:02:00 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 00:02:00 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 00:02:00 at 700 × g. Carefully remove and save the supernatant. Repeat this step twice, saving each supernatant fraction in a separate tube.
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.