TITLE: Nickel-NTA Protein Purification
AUTHORS: Nay Chi Khin, Diep Ganguly, Suyan Yee, Kai Xun Chan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Protocol to purify 6xHis-tagged recombinant proteins expressed in </span><span style = "font-style:italic;">E. coli</span><span> using Ni-NTA pull-down.</span></div></div>

[STEPS]
?. [Prepare buffers]
Buffer recipesResuspension buffer50 mM Tris-HCl, pH 8.02 mM EDTA1X Binding buffer 50 mM sodium phosphate buffer, pH 8.0 (ensure >1 pH units away from pI of expressed protein)500 mM sodium chloride0.5% Triton X-10010% glycerol10 mM imidazoleN.b. This should reflect the cell lysis buffer (e.g. https://www.protocols.io/private/3797D176057F0C37477116D259C75682).1X Wash buffer50 mM sodium phosphate buffer, pH 8.0500 mM sodium chloride0.5% Triton X-10010% glycerol20 mM imidazole1X Elute buffer50 mM sodium phosphate buffer, pH 8.0500 mM sodium chloride0.5% Triton X-10010% glycerol250 mM imidazoleN.b. DTT cleaves/interferes with Ni2+-binding, use low DTT concentrations (Digestion buffer 50 mM Tris-HCl, pH 8.0150 mM NaClFor storage, supplement digestion buffer with:1 mM DTT20% glycerol1X Binding buffer 50 mM sodium phosphate buffer, pH 8.0 (ensure >1 pH units away from pI of expressed protein)500 mM sodium chloride0.5% Triton X-10010% glycerol10 mM imidazoleN.b. This should reflect the cell lysis buffer (e.g. https://www.protocols.io/private/3797D176057F0C37477116D259C75682).1X Wash buffer50 mM sodium phosphate buffer, pH 8.0500 mM sodium chloride0.5% Triton X-10010% glycerol20 mM imidazole1X Elute buffer50 mM sodium phosphate buffer, pH 8.0500 mM sodium chloride0.5% Triton X-10010% glycerol250 mM imidazoleN.b. DTT cleaves/interferes with Ni2+-binding, use low DTT concentrations (Digestion buffer 50 mM Tris-HCl, pH 8.0150 mM NaClFor storage, supplement digestion buffer with:1 mM DTT20% glycerolN.b. This should reflect the cell lysis buffer (e.g. https://www.protocols.io/private/3797D176057F0C37477116D259C75682).1X Wash buffer50 mM sodium phosphate buffer, pH 8.0500 mM sodium chloride0.5% Triton X-10010% glycerol20 mM imidazole1X Elute buffer50 mM sodium phosphate buffer, pH 8.0500 mM sodium chloride0.5% Triton X-10010% glycerol250 mM imidazoleN.b. DTT cleaves/interferes with Ni2+-binding, use low DTT concentrations (Digestion buffer 50 mM Tris-HCl, pH 8.0150 mM NaClFor storage, supplement digestion buffer with:1 mM DTT20% glycerol1X Elute buffer50 mM sodium phosphate buffer, pH 8.0500 mM sodium chloride0.5% Triton X-10010% glycerol250 mM imidazoleN.b. DTT cleaves/interferes with Ni2+-binding, use low DTT concentrations (Digestion buffer 50 mM Tris-HCl, pH 8.0150 mM NaClFor storage, supplement digestion buffer with:1 mM DTT20% glycerolN.b. DTT cleaves/interferes with Ni2+-binding, use low DTT concentrations (Digestion buffer 50 mM Tris-HCl, pH 8.0150 mM NaClFor storage, supplement digestion buffer with:1 mM DTT20% glycerolFor storage, supplement digestion buffer with:1 mM DTT20% glycerol
?. [Ni-NTA Affinity Purification]
Keep solubilised protein samples (from completion of https://www.protocols.io/private/3797D176057F0C37477116D259C75682) on ice to thaw completely.
?. [Ni-NTA Affinity Purification]
Centrifuge samples (14,000 rcf for 10-20 minutes) and syringe filter (0.45 μm) supernatant into clean microfuge tube. Keep on ice.
?. [Ni-NTA Affinity Purification]
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.
?. [Ni-NTA Affinity Purification]
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).
?. [Ni-NTA Affinity Purification]
Keep column up-right and allow resin to settle. Once settled, open cap and capture flow-through (FT).
?. [Ni-NTA Affinity Purification]
Wash resin three-times with 1X Wash buffer (2x resin volume, i.e. 1 mL) and collect separately (i.e. W1, W2, W3).
?. [Ni-NTA Affinity Purification]
Elute four-times with 1X Elute buffer (1X resin volume, i.e. 500 μL), collect seperately (i.e. E1, E2, E3, E4).
?. [Ni-NTA Affinity Purification]
Run SDS-PAGE of pre-FT (20 μL), FT (20 μL), W1-W3 (20 μL), and E1-E4 (8 μL) to quality-check purification.
?. [Protease digestion]
Pool elutes containing protein fragment of expected size.
?. [Protease digestion]
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).
?. [Protease digestion]
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.
?. [Protease digestion]
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.
?. [Protease digestion]
Run elutes on SDS-PAGE gel to test for cleavage.
?. [Protease digestion]
Perform buffer exchange via centrifuge filtration (or dialysis) into Digestion buffer to reduce [imidazole] 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).
?. [Ni-NTA Affinity Purification]
Store all samples @ 4 ºC for the short-term. Keep columns containing resin for second Ni-NTA step.
?. [Protease digestion]
Prepare aliquots of purified protein supplemented with 10-20% glycerol and 1mM DTT. Store at -20 ºC.
?. [Protease digestion]
Pool and concentrate the pure fraction(s) using buffer exchange.