Jul 19, 2020
Nickel-NTA Protein Purification
- Nay Chi Khin1,
- Diep R Ganguly2,
- Suyan Yee1,
- Kai Xun Chan1
- 1The Australian National University;
- 2University of Pennsylvania
- Pogson Group
Protocol Citation: Nay Chi Khin, Diep R Ganguly, Suyan Yee, Kai Xun Chan 2020. Nickel-NTA Protein Purification. protocols.io https://dx.doi.org/10.17504/protocols.io.bihhkb36
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: Working
We use this protocol and it's working
Created: July 12, 2020
Last Modified: July 19, 2020
Protocol Integer ID: 39177
Keywords: Ni-NTA, Protein purification,
Abstract
Protocol to purify 6xHis-tagged recombinant proteins expressed in E. coli using Ni-NTA pull-down.
Guidelines
Try to perform all steps at 4 ºC as best as possible and keep protein samples on ice. For all spin-concentrate and buffer exchange steps, use slow speed (e.g. 3500-5000 rcf) to prevent precipitation.
Materials
MATERIALS
Sodium dihydrogen phosphateP212121
Disodium hydrogen phosphate
Triton-X100
1.5 mL Eppendorf tubes
GlycerolBio Basic Inc.Catalog #GB0232.SIZE.500ml
Poly-Prep Chromatography ColumnsBioRad SciencesCatalog #731-1550
DTTSigma AldrichCatalog #43816-10ML
Sodium chlorideSigma AldrichCatalog #S3014
ImidazoleSigma AldrichCatalog #I5513
Bovine Thrombin ProteinThermo FisherCatalog #1996000JL
HisPur™ Ni-NTA ResinThermo FisherCatalog #88221
Pierce™ HRV 3C Protease Solution Kit (2 units/µL)Thermo FisherCatalog #88946
TEV proteaseNew England BiolabsCatalog #P8112S
Tris Hydrochloride (Tris-HCl)Sigma AldrichCatalog #RES3098T-B7
Amicon® Ultra-4 Centrifugal Filter UnitMerck MilliporeCatalog #UFC8003
Amicon® Ultra-15 Centrifugal Filter UnitMerck MilliporeCatalog #UFC9003
Prepare buffers
Prepare buffers
1h
1h
Buffer recipes
Resuspension buffer
- 50 mM Tris-HCl, pH 8.0
- 2 mM EDTA
1X Binding buffer
- 50 mM sodium phosphate buffer, pH 8.0 (ensure >1 pH units away from pI of expressed protein)
- 500 mM sodium chloride
- 0.5% Triton X-100
- 10% glycerol
- 10 mM imidazole
N.b. This should reflect the cell lysis buffer (e.g. https://www.protocols.io/private/3797D176057F0C37477116D259C75682).
1X Wash buffer
- 50 mM sodium phosphate buffer, pH 8.0
- 500 mM sodium chloride
- 0.5% Triton X-100
- 10% glycerol
- 20 mM imidazole
1X Elute buffer
- 50 mM sodium phosphate buffer, pH 8.0
- 500 mM sodium chloride
- 0.5% Triton X-100
- 10% glycerol
- 250 mM imidazole
N.b. DTT cleaves/interferes with Ni2+-binding, use low DTT concentrations (<2 mM), in the above buffers, if needed.
Digestion buffer
- 50 mM Tris-HCl, pH 8.0
- 150 mM NaCl
For storage, supplement digestion buffer with:
- 1 mM DTT
- 20% glycerol
1h
Ni-NTA Affinity Purification
Ni-NTA Affinity Purification
35m
35m
Keep solubilised protein samples (from completion of https://www.protocols.io/private/3797D176057F0C37477116D259C75682) on ice to thaw completely.
15m
Centrifuge samples (14,000 rcf for 10-20 minutes) and syringe filter (0.45 μm) supernatant into clean microfuge tube. Keep on ice.
30m
Add an appropriate amount of Ni-NTA resin slurry (50% slurry in 20% ethanol) into gravity-flow column. For 15 mL bacterial cultures resuspended in 2 mL lysis buffer, equating to ~1-4 mg protein (1-2 mg/mL), 500 µL resin slurry is added.
Top-up column with 5x volume of 1X binding buffer (e.g. 500 µL slurry = 250 µL resin = ~2 mL 1X binding buffer).
Allow binding buffer to drop into a waste bottle to equilibrate the resin.
Transfer syringe filtered soluble protein (i.e. supernatant from step 3) into equilibrated resin, cap column tightly at both ends (including parafilm to prevent leakage) and incubate with shaking for 1-2 hours @ 4ºC (max 10 mL per column).
2h
Keep column up-right and allow resin to settle. Once settled, open cap and capture flow-through (FT).
Wash resin three-times with 1X Wash buffer (2x resin volume, i.e. 1 mL) and collect separately (i.e. W1, W2, W3).
Elute four-times with 1X Elute buffer (1X resin volume, i.e. 500 μL), collect seperately (i.e. E1, E2, E3, E4).
Run SDS-PAGE of pre-FT (20 μL), FT (20 μL), W1-W3 (20 μL), and E1-E4 (8 μL) to quality-check purification.
Store all samples @ 4 ºC for the short-term. Keep columns containing resin for second Ni-NTA step.
Protease digestion
Protease digestion
16h
16h
Pool elutes containing protein fragment of expected size.
Perform buffer exchange via centrifuge filtration (or dialysis) into Digestion buffer to reduce [imidazole] < 1mM.
For example, if 1X Elution buffer contains 250 mM imidazole, you will need 4x centrifugation at a dilution of 1:4 per spin (4 ºC @ 3,500 rcf for 20 minutes).
Add appropriate protease (e.g. TEV, Thrombin, HRV 3C) and incubate overnight (or > 16hrs) at 4 ºC. Ensure to add any required cofactors (e.g. citrate for TEV).
16h
Equilibrate Ni-NTA resin (from previously used and stored columns) with 10-20 mL Digestion buffer and allow resin to settle. Let buffer flow-through.
Add digested protein to resin and collect flow-through (dig-FT). Can perform multiple elutes with Digestion buffer to obtain as much protein as possible.
Run elutes on SDS-PAGE gel to test for cleavage.
Pool and concentrate the pure fraction(s) using buffer exchange.
Prepare aliquots of purified protein supplemented with 10-20% glycerol and 1mM DTT. Store at -20 ºC.