Ni NTA Beads 6FF
Ni NTA Beads 6FF is based on a highly cross-linked 6% agarose with the same ligand as Ni NTA Beads (See Fig 1.), see table 1. In addition to being able to tolerate harsh reagent conditions (see Table 2), Ni NTA Beads 6FF is more stable due to its pressure-resistant matrix, which can withstand pressures up to 0.3 MPa, making the product is more suitable for industrial large-scale protein purification, which can be achieved at relatively high flow rates for the purification of target proteins.
Fig.1. The chemical structures of Ni IDA Beads, Ni NTA Beads and Ni NTA Beads 6FF
Table 1. Characteristics of Ni NTA Beads 6FF
Item | Description |
Matrix Spherical | Highly cross-linked 6% agarose |
Static Binding Capacity | ＞40 mg 6×His-tagged protein/ml medium |
Particle size | 45-165 μm |
Maximum Pressure | 0.3 MPa, 3 bar |
Storage Solution | 1×PBS containing 20% ethanol |
Storage Temperature | 4-30℃ |
Table 2. Chemical compatibilities for Ni NTA Beads 6FF
Reagent | Stability |
Reductants | 5 mM DTE 1mM DTT 20 mM β-mercaptoethanol 5 mM TCEP 10 mM reduced glutathione |
Denaturants | 8 M urea 6 M Gua-HCl |
Table 2. Chemical compatibilities for Ni NTA Beads 6FF (Continued table)
Reagent | Stability |
Detergent | 2% Triton X-100 (nonionic) 2% Tween 20 (nonionic) 2% NP-40 (nonionic) 2% cholate (anionic) 1% CHAPS (zwitterionic) |
Other additives | 500 mM imidazole 20% ethanol 50% glycerol 100 mM Na2SO4 1.5 M NaCl 1 mM EDTA 60 mM citrate |
2.1 Buffer Preparation
The basic principle of the following recommended buffer and other buffer is low concentration of imidazole in Lysis and wash buffer and high in elution buffer. Water and chemicals used for buffer preparation should be of high purity. It is recommended to filter the buffers by passing them through a 0.22 μm or 0.45 μm filter before use. Ni NTA Beads 6FF can be used for the his-tagged protein purification under native conditions and denaturing conditions, which need different buffer. See table 3, table 4 and table 5.
Table 3. Recommended buffer for his-tagged protein purification under native conditions
Name | Volume | Ingredient |
Lysis Buffer | 1 L | 50 mM NaH2PO4 (7.80 g NaH2PO4·2H2O) 300 mM NaCl (17.54 g NaCl) 10 mM imidazole (0.68 g imidazole) Adjust the buffer pH to 8.0 with NaOH solution. |
Wash Buffer | 1 L | 50 mM NaH2PO4 (7.80 g NaH2PO4·2H2O) 300 mM NaCl (17.54 g NaCl) 20 mM imidazole (1.36 g imidazole) Adjust the buffer pH to 8.0 with NaOH solution. |
Elution Buffer | 1 L | 50 mM NaH2PO4 (7.80 g NaH2PO4·2H2O) 300 mM NaCl (17.54 g NaCl) 250 mM imidazole (17.0 g imidazole) Adjust the buffer pH to 8.0 with NaOH solution. |
Table 4. Recommended buffer for his-tagged protein purification under denaturing conditions, pH elution
Name | Volume | Ingredient |
Lysis Buffer | 1 L | 8 M Urea (480.50 g Urea) 100 mM NaH2PO4 (15.60 g NaH2PO4·2H2O) 100 mM Tris·HCl (15.76 g Tris·HCl) Adjust the buffer pH to 8.0 with HCl solution |
Wash Buffer | 1 L | 8 M Urea (480.50 g Urea) 100 mM NaH2PO4 (15.60 g NaH2PO4·2H2O) 100 mM Tris·HCl (15.76 g Tris·HCl) Adjust the buffer pH to 6.3 with HCl solution |
Elution Buffer | 1 L | 8 M Urea (480.50 g Urea) 100 mM NaH2PO4 (15.60 g NaH2PO4·2H2O) 100 mM Tris·HCl (15.76 g Tris·HCl) Adjust the buffer pH to 4.5 with HCl solution |
Table 5. Recommended buffer for his-tagged protein purification under denaturing conditions, imidazole elution
Name | Volume | Ingredient |
Lysis Buffer | 1 L | 8 M Urea (480.50 g Urea) 50 mM NaH2PO4 (7.80 g NaH2PO4·2H2O) 300 mM NaCl (17.54 g NaCl) 10 mM imidazole (0.68 g imidazole) Adjust the buffer pH to 8.0 with NaOH solution. |
Wash Buffer | 1 L | 8 M Urea (480.50 g Urea) 50 mM NaH2PO4 (7.80 g NaH2PO4·2H2O) 300 mM NaCl (17.54 g NaCl) 20 mM imidazole (1.36 g imidazole) Adjust the buffer pH to 8.0 with NaOH solution. |
Elution Buffer | 1 L | 8 M Urea (480.50 g Urea) 50 mM NaH2PO4 (7.80 g NaH2PO4·2H2O) 300 mM NaCl (17.54 g NaCl) 250 mM imidazole (17.0 g imidazole) Adjust the buffer pH to 8.0 with NaOH solution. |
Note: 8 M Urea could also be replaced by 6 M Guanidine-Hcl. Table 4 or Table 5 select one of them as the solution for the inclusion body purification.
2.2 Sample Preparation
2.2.1 Recombinant native protein expressed in E.coli or yeast
1) Single colonies were cultured in LB medium. According to the instruction, adding the inducers for a period of time.
2) Harvest cells from an appropriate volume of bacterial culture by centrifugation at 7,000rpm (7,500×g) for 10-15min at 4℃. Discard supernatant and determine weight of pellet. Resuspend pellet in 1:10 ration (w/v) in lysis buffer and add lysozyme (0.2-0.4 mg/ml cell paste, if the host cell containing pLysS or pLysE, it can be without lysozyme) and PMSF (1 mM/ml cell paste).
3) If the concentration of cell suspension is higher, it is recommended to add 10μg/ml RNase A and 5μg/ml DNase I. Sonicate the cell suspension/lysate on ice.
4) Centrifuge the homogenized lysate at 10,000 rpm for 20 to 30 min at 4℃ to clarify sample. Save supernatant.
2.2.2 Native protein expressed in yeast, insect or mammalian cells
1) Harvest the cells from an appropriate volume of culture by centrifugation at 5,000 rpm for 10-15 min at 4℃. Save supernatant. If the supernatant without EDTA, histidine and reductant, it can be purified directly, otherwise it need dialysis to 1×PBS under 4℃.
2) For a large volume of supernatant, it need precipitation by adding ammonium sulfate and dialysis to 1×PBS under 4℃.
2.2.3 Inclusion bodies from E.coli
1) Harvest cells from an appropriate volume of bacterial culture by centrifugation at 7,000 rpm for 10-15min at 4℃. Discard supernatant and determine weight of pellet.
2) Resuspend pellet in 1:10 ration (w/v) with Lysis Buffer. Sonicate the cell suspension/lysate on ice.
3) Centrifuge the homogenized sample at 10,000 rpm or 20 min at 4℃ to pellet the inclusion.
4) Resuspend pellet in 1:10 ration (w/v) with denaturing Lysis Buffer (containing 8 M urea). Sonicate, as needed, to redissolve the pellet.
5) Analyze the concentration of the target protein and continue with purification protocols under denaturing conditions.
2.3 Packing Columns
Ni NTA Beads 6FF is easy to pack and use, and its high flow properties make it excellent for industrial scaling-up. The method of packing the column is described below.
1) Remove air from the column dead spaces by flushing the end-piece and adapter with packing buffer. Make sure no air has been trapped under the column net.
2) Close the column outlet leaving the net covered with packing buffer.
3) Resuspend the beads stored in its container by shaking (avoid stirring the sedimented medium). Pouring the slurry down a glass rod held against the column wall will minimize the introduction of air bubbles. If using a packing reservoir, immediately fill the remainder of the column and reservoir with packing buffer. Mount the adapter or lid of the packing reservoir and connect the column to a pump. Avoid trapping air bubbles under the adapter or in the inlet tubing.
4) Open the bottom outlet of the column and set the pump to run at the desired flow velocity. Ideally, Ni NTA Beads 6FF is packed at a constant pressure of approximately 3 bar (0.3 MPa). Initially the buffer should be allowed to flow slowly through the chromatography column and then slowly increase to the final flow rate. This will avoid the impact of hydraulic pressure on the formed column bed and will also avoid uneven formation of the column bed. If the recommended pressure or flow velocity can not be obtained, use the maximum flow velocity the pump can deliver. This should also give a reasonable well-packed bed. Do not exceed 75% of the packing flow velocity in subsequent chromatographic procedures.
5) Maintain packing flow velocity for at least 3 bed volumes. When the bed has stabilized, mark the bed height on the column and close the bottom outlet and stop the pump. If using a packing reservoir, disconnect the reservoir and fit the adapter to the column. If using the column, carefully place the top filter on top of the bed before fitting the adapter.
7) Connect the pump, open the bottom outlet and continue packing. The bed will be further compressed at this point and a space will be formed between the bed surface and the adapter.
8) Close the bottom outlet. Disconnect the column inlet and lower the adapter approximately 2 mm into the bed. Connect the pump. The column is now ready to use.
2.4 Sample Purification
1) Fill the syringe or pump tubing with distilled water. Remove the stopper and connect the column to the syringe (with the provided connector), or pump tubing,“drop to drop”to avoid introducing air into the column. Remove the snap-off end at the column outlet.
2) Wash the column with 3-5 column volumes of distilled water.
3) Equilibrate the column with at least 5 column volumes Lysis Buffer.
4) Load the pre-treated sample by using a Loop fitted to the connector or by pumping it onto the column.
5) Wash the column with10-15 column volumes of wash buffer or until the absorbance reaches the baseline or no material appears.
6) Elute with elution buffer using a stepwise or linear gradient. For one-step elution, 5 column volumes are usually enough. Other volumes may be required if the interaction is difficult to break. Linear gradient elution can be used to separate proteins of different binding strengths with a small gradient, such as 20 column volumes or more.
2.5 Analysis
Identify the fractions containing the His-tagged protein. Use UV absorbance, SDS-PAGE, or western blot.
A column used to purify protein from cell extract usually contains some soluble substances and cell debris that are nonspecifically absorbed onto the matrix.
Cleaning-in-Place eliminates material not removed by regeneration and prevents progressive buildup of contaminants. If the column is to be reused, these contaminants should be cleaned from the column, as they were not completely removed during the sample clarification steps.
Remove the strong hydrophobic binding protein, lipoprotein and lipid
Wash the column with 5-10 column volumes 30% isopropanol contacting for 15-20 min. Or you can choose the 2 column volumes acidic or alkaline solution containing detergents, for example, 0.1 M acetic acid solution contains 0.1-0.5% non-ionic detergent, contacting for 1-2 hours .
Finally wash the column with 10 column volumes distilled water.
Remove the proteins combined with ion interacting
Wash the column with 1.5 M NaCl solution contacting for 10-15 min.
Finally wash the column with 10 column volumes distilled water.
In general, Ni NTA Beads 6FF may be used a number of times before it becomes necessary to recharge them with metal ions. When the back pressure is too high or the capacity significantly lower, it need to strip the metal ions and recharge the Ni NTA Beads 6FF as the following procedure.
Wash the column with one of the following solutions.
1) Rinse with 5 column volumes of distilled water；
2) 100 mM EDTA (pH 8.0), 5 column volumes；
3) Rinse with 10 column volumes of distilled water；
4) 0.5 M NaOH, 5 column volumes, contacting for 10-15 min；
5) Rinse with 10 column volumes of distilled water；
6) 100 mM NiSO4, 3-5 column volumes；
7) Rinse with 10 column volumes of distilled water；
After regeneration, the medium can be used immediately, otherwise, it need to be suspended in an equal volume of 1×PBS containing 20% ethanol at 4℃.
Problem | Probable Cause | Solution |
Back pressure exceeds 3 bar | Column is clogged | Cleaning in place (part 3). |
Increase the centrifugation speed or filtering the sample. | ||
Sample is too viscous | Increase sonication or add DNase I (5 μg/ml with 1mM Mg2+. Incubate on ice for 15 min. | |
Dilute sample with Binding buffer. | ||
No protein is eluted | Protein may be an inclusion body, not in the supernatant. | The lysate can be analyzed by electrophoresis for the presence of the target protein in the supernatant, and the inclusion body protein need to be purified according to the purification method of the inclusion body protein. |
Expression of target protein in extract is very low | Check expression level of protein by estimating the amount in the extract, flow through, elute fraction and pellet upon centrifugation. Or apply large sample volume. | |
Target protein is found in the flow through | Reduce imidazole concentration in sample lysis and wash buffer. Or increase the pH of buffer. | |
Protein degradation or purification cause the histidine tag to be removed. | Operate at 4℃. Add protease inhibitors. | |
Make a new construct with his-tag attached to other terminus. | ||
His-tagged protein is not pure | Wash is not enough | Increase the volume of Wash Buffer. |
Association between the his tagged protein and protein contaminant. | Optimize the wash condition by adjusting the pH and imidazole concentration. | |
Add an additional chromatography step, that is ion exchange, hydrophobic interaction or size exclusion. | ||
The color of medium becomes shallow. | The nickel ions was stripped. | Chelate nickel ions according to the part 4. |
The color of medium becomes brown | The buffer contains reducing agent. | Refer to Table 2 to reduce the concentration of reducing agent |
Protein precipitates during purification | Temperature is too high | Perform the purification at 4℃. |
Aggregate formation | Add solubilization agents to the samples and buffers, for example 0.1% Triton X-100 , Tween-20 and ≤20% glycerol to maintain protein solubility. |
Product | Cat. No. | Size |
Ni NTA Beads 6FF | SA005005 SA005025 SA005100 SA005500 SA00501L SA00510L | 5 ml 25 ml 100 ml 500 ml 1 L 10 L |
