Ni-NTA agarose gel 6FF is a purification medium for the purification of 6×His labeled recombinant proteins, which is made from 6% cross-linked Sepharose coupled with a tetradentate chelating agent NTA. It can be used to purify 6×His labeled recombinant proteins expressed by any expression system under non-denaturing or denaturing conditions. NTA, which contains four chelating zones, binds Ni2+ better than the common three-tooth chelating agent. 6×His can be chelated with Ni2+, so that His label protein is bound on the Ni-NTA purification medium, the unbound protein is washed off, and the protein bound on the medium is gently eluted by a certain concentration of imidazole or low pH buffer, so as to obtain a high purity of the target protein. The purification medium has a very high affinity with His label proteins, up to 5-20 mg/ml. His tag proteins derived from any expression system can be purified under both non-denaturing and denaturing conditions. The purification procedure is simple and the resulting protein purity can be as high as 95%. Ni-NTA can be regenerated 4-6 times and reused. The suspension of this product is 20% ethanol, chelated Ni2+.

Operation Method:

A. Extraction of His label protein under non-denaturing conditions

1) Prepare cells, inoculate and induce expression. Collect the cells and place them at -70℃or proceed to step 2 immediately.

2) NTA-0 Buffer and PMSF of 1/20 cell growth volume were added. The working concentration used by PMSF is 1 mM.

3) Suspend the cells, add lysozyme, mix well, place on ice for 30 minutes, ultrasound or homogenate to break the cells. This step is performed on ice.

4) Add 10% Triton X-100 to a final concentration of 0.05%, mix thoroughly and leave on ice for 15 minutes.

5) At 12000 rpm/min(above 20,000×g), centrifuge at 4℃for more than 15 minutes. Remove supernatant and place on ice for reserve or store at -20℃.

6) The NTA resin was loaded into a suitable chromatographic column, and the chromatography was washed with NTA-0 Buffer of 10 times the volume of NTA.

7) The sample was added to the NTA chromatographic column at a flow rate of about 15 ml/h. The penetrating part was collected and the protein binding was analyzed by SDS/PAGE.

8) The chromatography was washed with NTA-0 Buffer of 5 times the volume of NTA, and the flow rate was controlled at about 30 ml/h.

9) Eluent was eluted with NTA-20, NTA-40, NTA-60, NTA-80, NTA-100, NTA-200, and NTA-1000 Buffer of 5 times the volume of NTA respectively, and the flow rate was controlled at about 15 ml/h. The eluent was collected, and one NTA volume was collected in each tube.

10) Determine the distribution of the target protein in the eluent. The most effective method is SDS-PAGE analysis. You can also use the Bradford protein assay kit to quickly determine the protein content and then analyze the protein distribution with SDS/PAGE.

11) The preservation conditions of the purified target protein need to be determined according to the nature and use of the protein. NTA-0, 20, 40, 60, 80, 100, 200, 1000Buffer showed a concentration of 20 mM Tris-HCl pH7.9, 0.5 M NaCl, and 10% Glycerol, respectively. Add Imidazole of 0, 20, 40, 60, 80, 100, 200, and 1000 mM.

B. Purification of His label proteins from inclusion bodies under denaturation conditions

1) Prepare cells, inoculate and induce expression. Collect the cells and place them at -70℃or proceed to step 2 immediately.

2) Add GuNTA-0 Buffer and PMSF of 1/20 cell growth volume. The working concentration used by PMSF is 1 mM.

3) The cells are suspended, and the cells are broken by ultrasound on ice to reduce the viscosity.

4) Leave at room temperature for 30 minutes, occasionally mix or stir with magnetic force.

5) At 12000 RPM (above 20,000×g), centrifuge at 4℃for more than 15 minutes. Remove supernatant and place on ice for reserve or store at -20℃.

6) NTA resin was loaded into a suitable chromatographic column, and the chromatography was washed with GuNTA-0 Buffer 10 times the volume of NTA.

7) The sample was added to the NTA chromatographic column, the flow rate was controlled at about 15 ml/h, and the penetrating part was collected for SDS/PAGE analysis of protein binding.

8) The chromatography was washed with 5 times the volume of NTA GuNTA-0 Buffer, and the flow rate was controlled at about 30 ml/h.

9) Elution with 5 times NTA volume GuNTA-20, GuNTA-40, GuNTA-60, GuNTA-100, GuNTA-500, respectively, at the flow rate of about 15 ml/ h, collect the eluent, and collect one NTA volume per tube.

10) Determine the distribution of the target protein in the eluent. The most effective method is SDS/PAGE analysis. You can also use the Bradford protein assay kit to quickly determine the protein content and then analyze the protein distribution with SDS/PAGE.

11) The target protein needs to be further purified according to the purpose of the protein.

12) The purified target protein needs to be renaturated, and the common means of renaturation can be determined by referring to the principles of the relevant manual.

The concentration of GuNTA-0, 20, 40, 60, 100 and 500Buffer is 20 mM Tris-HCl pH7.9 and 0.5 M NaCl, respectively. 10% Glycerol,6 M Guanidium HCl were added with 0, 20, 40, 60, 100, 500 mM Imidazole.

Regeneration of C.NTA resin

The binding efficiency of NTA resin decreases after several times of use (3-5 times), and it can be regenerated by the following methods to improve the service life of the resin and the binding efficiency of the protein.

NTA resin needs to drain all the solution from the lower end of the chromatographic column before re-creation, estimate the resin volume of NTA, add the regeneration reagent to the chromatographic column in the following order, wait for the regeneration solution of the previous step to dry, and then add the next step of regeneration dissolution.

Users need to prepare their own 25%, 50%, 75%, 100% (v/v) ethanol and deionized water. Drain all solution from the bottom of the chromatographic column, Stripping Solution I with twice the volume of NTA resin, twice the volume of deionized water, and three times the volume of Stripping Solution II, 1 volume 25% ethanol, 1 volume 50% ethanol, 1 volume 75% ethanol, 5 volume 100% ethanol, 1 volume 75% ethanol, 1 volume 50% ethanol, 1 volume 25% ethanol, 1 volume deionized water, 5 volume Stripping Solution III, 3 times the volume of deionized water washed again.

If used immediately, wash with 5 times the volume of Ni Charging Solution and then 10 times the volume of balance solution (NTA-0Buffer or GuNTA-0 Buffer). For long-term storage, add 1 times the volume of 20% ethanol, store at 4℃, wash with 5 times the volume of Ni Charging Solution before use, and then wash with 10 times the volume of equilibrium solution (NTA-0Buffer or GuNTA-0 Buffer)

Regenerated Solution formula: Stripping Solution I: 6M GuHCl, 0.2M acetic acid. Stripping Solution II: 2% SDS. Stripping Solution III: 100 mM EDTA, pH 8.0. Ni Charging Solution: 100 mM NiSO4

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