Ni-NTA purification of denatured His-tagged nanoCLAMP proteins from NEc1 cell pellets


nanoCLAMPs with an N-terminal His-tag and a C-terminal cysteine are purified from BL21-derivative NEc1 under denaturing conditions with Ni-NTA resin. 


1 Prepare "Denaturing IMAC Buffer - TCEP"
Make 10 ml per g of cell pellet plus safety
2 Add 10 ml "Denaturing IMAC Buffer - TCEP" per g of cell pellet and resuspend
The cell pellet is prepared as described in the protocol "Induction of Protein Expression from E. coli NEc-1 - Frozen Stock to Cell Pellet"
3 Sonicate with a homogenizing tip until the cell pellet is fully resuspended
Typically 2 to 3 minutes. Minimize foaming. Example setup: Kinematica Polytron PT-10-35 with tip PTA 10 TS, level <3
4 Transfer the suspension to a 30 ml Oak Ridge Centrifuge Tubes
Transfer up to 18 ml of suspension per tube to avoid leaking
5 Centrifuge the suspension in a fixed angle rotor at 30,000 X g for 30 minutes at 15°C
Thermo FiberLite F21-8x50y at 15,900 rpm
6 During the centrifugation step, transfer the appropriate volume of "Qiagen Ni-NTA resin, 50% slurry" to a 50 ml conical tube
The appropriate volume of resin typically ranges from 0.6 to 1.4 ml per g of cell pellet. The appropriate amount can be determined by small scale titration.
7 Centrifuge the Ni-NTA resin slurry at 1000 X g for 4 minutes
Beckman S4180 at 2,448 rpm
8 Discard the supernatant, resuspend the resin in 3 bed volumes of "Denaturing IMAC Buffer," and centrifuge in a swinging bucket rotor at 1000 X g. Repeat this step for a total of 3 times
9 Resuspend the Ni-NTA resin in "Denaturing IMAC Buffer" to make a 50% slurry and then transfer the slurry to a conical tube or chromatography column. Drain or centrifuge to remove excess buffer
Use a container with sufficient volume for the resin and the cleared lysate
10 Transfer supernatant from the centrifugation of the cell suspension (cleared lysate) to the container containing the Ni-NTA resin
11 Seal the container of cleared lysate and Ni-NTA resin and rotate for at least for 2 hours at Room Temperature
Binding time can be increased to up to 24 hours
12 Remove the cleared lysate either by draining the column or centrifuging at 1000 X g for 4 minutes
If desired, save the supernatant for analysis
13 If the Ni-NTA resin is in a centrifuge tube, add "Denaturing IMAC Buffer - TCEP" to form a slurry. Transfer the slurry to a chromatography column. Wash the centrifuge tube with additional buffer and transfer the wash to the column to maximize recovery
14 Connect the column to a liquid chromatography system with a flow adapter filled with "Denaturing IMAC Buffer"
15 Wash the column with 3 bed volumes of "Denaturing IMAC Buffer - TCEP"
For a 1 cm diameter column, set the flow rate at 1 ml/min. For a 2.5 cm diameter column, set the flow rate at 2 ml/min.
16 Wash the column with an additional 3 bed volumes of "Denaturing IMAC Buffer - TCEP" or until the A280 flatlines, whichever occurs first
17 Elute protein with "Denaturing IMAC Buffer - 250 mM Imidazole" and collect peak fractions
18 Store the protein protected from light for up to two weeks at Room Temperature

Solution(s) Used
Use / Nickname Solution Usage Rate
For lysing the cell pellets Denaturing IMAC Buffer - TCEP 10 ml per g of cell pellet
For equilibrating the Ni-NTA resin Denaturing IMAC Buffer 10 ml per ml of Ni-NTA resin
For transferring the Ni-NTA resin with bound protein and washing the column Denaturing IMAC Buffer - TCEP 10 ml per ml of Ni-NTA resin
For eluting the bound protein from the Ni-NTA resin Denaturing IMAC Buffer - 250 mM Imidazole 5 ml per ml of Ni-NTA resin

Ingredient(s) Used
Use / Nickname Ingredient Usage Rate
For "Denaturing IMAC Buffer - TCEP" TCEP HCl, 0.5 M 200 ul per 100 ml buffer

Outside Material(s) Used
Vendor Outside Material Catalog Number Usage Rate
Qiagen Ni-NTA Agarose - Max Pressure 2.8 psi 30230 1 ml of packed resin per 0.7 to 1.7 g of cell pellet
ThermoFisher Scientific Oak Ridge High Speed Polycarbonate Centrifuge Tube - 30 ml 3139 1 tube per 1.6 g of cell pellet