BioReagent, 50% v/v BioReagent for sensitive chromatographic and analytical workflows requiring minimal baseline interference.
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Co-NTA Affinity Resin 6FF (His tag) enables one‑step purification of His‑tagged proteins from a variety of expression systems, such as E. coli, yeast, insect cells, and mammalian cells. The product is based on highly cross‑linked 6% agarose gel as the matrix, with cobalt ions chelated via tetra‑dentate coordination to the ligand. Owing to its pressure‑resistant matrix, it can withstand up to 0.3 MPa, making it particularly suitable for large‑scale industrial protein purification. Co-NTA Affinity Resin 6FF (His tag) also tolerates a range of denaturants and other additives (see Table 2). Another feature of Co-NTA Affinity Resin 6FF (His tag) is its higher selectivity for His‑tagged proteins compared to Ni-NTA Affinity Resin 6FF (His tag).
Affinity Resin 6FF (His tag) is supplied as a 1:1 (v/v) slurry in 1× PBS containing 20% ethanol; the specified product volume refers to the actual settled gel volume.
Table 1. Performance properties of Co-NTA Affinity Resin 6FF (His tag)
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Table 2. Reagent tolerance of Co-NTA Affinity Resin 6FF (His tag)
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Notes:
① Do not store the medium in buffers containing β‑mercaptoethanol.
② Excessively high imidazole concentrations may reduce protein recovery.
③ Ethanol may cause protein precipitation, reduce recovery, and clog the column.
2. Purification Protocol
2.1 Buffer Preparation
The following recommended buffers can be used, or you may prepare different buffer systems according to your own preferences. The basic principle is loading at low imidazole concentration and eluting at high imidazole concentration. A neutral to slightly alkaline buffer (pH 7–8), such as phosphate buffer, is recommended. 0.3–0.5 M NaCl may be added to reduce non‑specific binding. Avoid using Tris‑HCl under conditions where protein binding is weak. Chelating agents such as EDTA and citrate buffers should be avoided. It is best to filter the buffers through a 0.22 μm or 0.45 μm membrane before use. Since Co-NTA Affinity Resin 6FF (His tag) can be used for both soluble protein and inclusion body protein purification, the buffers required differ between the two methods. Specific formulations are given in Table 3 and Table 4.
Table 3. Buffers and formulations for soluble His‑tagged protein purification
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Table 4. Buffers and formulations for inclusion‑body His‑tagged protein purification (imidazole elution)
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2.2 Sample Preparation
2.2.1 Proteins expressed in bacteria or yeast
1.Pick a single colony into culture medium. Add the appropriate concentration of inducer according to the vector instructions and induce for the appropriate time.
2.After expression, transfer the culture to centrifuge bottles. Centrifuge at 7,000 rpm (7,500×g) for 15 min to collect the cells. Resuspend the cell pellet in Lysis Buffer at a ratio of 1:10 (W/V). Add PMSF to a final concentration of 1 mM. Add lysozyme (working concentration 0.2–0.4 mg/mL; if the host cells contain pLysS or pLysE, lysozyme may be omitted). Other protease inhibitors may also be added, provided they do not affect binding of the target protein to the medium.
3.Resuspend the cell pellet (if the cell density is high, consider adding 10 μg/mL RNase A and 5 μg/mL DNase I), mix well, and place on ice. Sonicate on ice until the cell suspension becomes essentially clear.
4.Transfer the clarified lysate to centrifuge tubes. Centrifuge at 10,000 rpm (15,000×g) for 20–30 min at 4 °C. Collect the supernatant and keep on ice for immediate use or store at –20 °C.
2.2.2 Soluble proteins secreted by yeast, insect, and mammalian cells
1.Transfer the cell culture medium to centrifuge cups and centrifuge at 5,000 rpm (3,800×g) for 10 min to remove cells. If the supernatant does not contain EDTA, histidine, reducing agents, or similar substances, it can be applied directly to the column. If it contains such substances, dialyze against Lysis Buffer before loading.
2.For large volumes of supernatant, concentrate by ammonium sulfate precipitation; the protein must then be dialyzed against Lysis Buffer before loading onto the column.
2.2.3 Inclusion body protein purification (denaturing conditions)
1.Transfer the culture to centrifuge bottles and centrifuge at 7,000 rpm (7,500×g) for 15 min to collect the cells. Discard the supernatant.
2.Resuspend the cells in Lysis Buffer (without 8 M urea) at a ratio of 1:10 (W/V), mix well, and sonicate on ice.
3.Transfer the lysate to centrifuge tubes and centrifuge at 10,000 rpm (15,000×g) for 20–30 min at 4 °C. Discard the supernatant. Steps 2 and 3 may be repeated once.
4.Resuspend the inclusion bodies in Lysis Buffer (containing 8 M urea) at a ratio of 1:10 (W/V).
5.Purify the His‑tagged protein under denaturing conditions using the buffer formulations given in Table 4.
2.3 Packing of Co-NTA Affinity Resin 6FF (His tag)
2.3.1 Packing a gravity column
1.Take a gravity column of suitable size, insert the lower frit, rinse the column tube and frit with an appropriate amount of pure water, and close the bottom outlet.
2.Mix the Co-NTA Affinity Resin 6FF (His tag) thoroughly. Using a pipette, transfer an appropriate volume of slurry into the gravity column (the actual settled medium volume is half of the slurry volume). Open the bottom outlet and allow the storage solution to drain.
3.Add an appropriate amount of pure water to wash the medium. After the liquid has drained by gravity, close the bottom outlet.
4.Insert the rinsed upper frit, ensuring that there is no gap between the frit and the resin bed and that the frit is level.
5.The packed gravity column can be equilibrated directly with equilibration buffer. If not used immediately, add storage solution and store at 2–8 °C.
2.3.2 Packing a medium‑pressure chromatography column
Co-NTA Affinity Resin 6FF (His tag) is widely used in industrial purification and is therefore packed into various medium‑pressure chromatography columns. The packing method is described below. Before packing, calculate the column base area from the column diameter, and calculate the required medium volume based on the desired packed bed height using the following formula:
V = 1.15 × π × r² × h
(V: required medium volume in mL; 1.15: compression factor; r: column radius in cm; h: packed bed height in cm)
Note: The volume of slurry taken should be twice the medium volume, because the settled medium accounts for only half of the total slurry volume; the other half is storage solution.
1.Rinse the column bottom frit and adapter with deionized water, ensure no air bubbles remain on the bottom frit, close the bottom outlet, and leave 1–2 cm of deionized water in the column bottom.
2.Resuspend the medium and carefully pour the slurry continuously into the column. Pouring the slurry along the column wall with a glass rod can help reduce bubble formation.
3.If a reservoir is used, immediately fill the column and the reservoir with water. Place the flow adapter on the slurry surface, connect to the pump, and avoid introducing air bubbles into the adapter or tubing.
4.Open the outlet at the bottom of the chromatography column and start the pump to run at the set flow rate. Initially, pass the buffer through the column at a low flow rate, then gradually ramp up to the final flow rate. This prevents hydraulic shock to the packed bed and avoids uneven bed formation.If the recommended pressure or flow rate cannot be reached, the maximum flow rate of your pump may be used, which can still give good packing results. (Note: In subsequent chromatography runs, do not exceed 75% of the maximum packing flow rate.) Once the bed height is stable, pass at least 3 column volumes of deionized water through the bed at the final packing flow rate. Mark the bed height.
5.Stop the pump and close the column outlet.
6.If a reservoir was used, remove it and place the adapter into the column.
7.Push the adapter down to the marked bed height. Allow packing liquid to enter the adapter, then lock the adapter fitting.
8.Connect the packed column to a pump or chromatography system and begin equilibration. The adapter can be readjusted if necessary.
2.4 Sample Purification Procedures
2.4.1 Batch incubation purification
1.According to the sample volume, transfer an appropriate amount of Co-NTA Affinity Resin 6FF (His tag) to a centrifuge tube. Centrifuge at 1,000 rpm for 1 min and aspirate the supernatant. Alternatively, use a gravity column and allow the storage solution to drain.
2.Add 5 medium volumes of Lysis Buffer to the centrifuge tube to wash the medium. Centrifuge at 1,000 rpm for 1 min and aspirate the supernatant. If using a gravity column, wash directly in the column by gravity flow. Repeat at least twice.
3.Add the sample, close the centrifuge tube or gravity column, and incubate with shaking at 4 °C for 2–4 h, or at 37 °C for 30 min–2 h.
4.After incubation, centrifuge at 1,000 rpm for 1 min and aspirate the supernatant, or filter to collect the medium. Retain the supernatant as the flow‑through fraction for electrophoretic analysis.
5.Wash the medium with 5 medium volumes of Wash Buffer. Centrifuge at 1,000 rpm for 1 min or filter through the gravity column, and remove the supernatant (be careful not to aspirate the medium). Repeat 3–5 times; it is recommended to transfer to a new centrifuge tube in between washes. The imidazole concentration in the wash buffer may be adjusted if necessary.
6.Add 3–5 column volumes of Elution Buffer and incubate at room temperature for 10–15 min. Centrifuge at 1,000 rpm for 1 min or collect the eluate from the gravity column. This elution step can be repeated 2–3 times.
2.4.2 Gravity column purification
1.Equilibrate the packed Co-NTA Affinity Resin 6FF (His tag) gravity column with 5 column volumes of Lysis Buffer to bring the resin into the same buffer system as the target protein. Repeat 2–3 times.
2.Apply the sample to the equilibrated gravity column. Allow a residence time of at least 2 min to ensure sufficient contact between the sample and the medium. Collect the flow‑through. The sample can be loaded repeatedly to increase binding efficiency.
3Wash with 10–15 column volumes of Wash Buffer to remove non‑specifically bound contaminating proteins. Collect the wash fractions. The imidazole concentration in the wash buffer may be adjusted if necessary.
4.Elute with 5–10 column volumes of Elution Buffer. Collect fractions in separate tubes (one column volume per tube) and analyze each. This ensures that all bound target protein is eluted while also yielding protein of high purity and concentration.
2.4.3 Medium‑pressure column purification
After packing, Co-NTA Affinity Resin 6FF (His tag) can be used with various conventional medium‑ and low‑pressure chromatography systems.
1.Fill the pump tubing with deionized water. Remove the top cap, connect the column to the chromatography system, open the bottom outlet, attach the prepacked column, and tighten the connections.
2.Wash out the storage buffer with 3–5 column volumes of deionized water.
3.Equilibrate the column with at least 5 bed volumes of Lysis Buffer.
4.Load the sample using the pump or sample loop. Note: Increased sample viscosity can cause high backpressure even with small injection volumes. Do not exceed the binding capacity of the column. Large sample volumes may also cause high backpressure, making the injector more difficult to use.
5.Wash the column with Wash Buffer until the UV absorbance reaches a stable baseline (generally at least 10–15 column volumes). Note: Adding imidazole to the sample and binding buffer can improve sample purity.
6.Elute using Elution Buffer in either stepwise or linear gradient mode. For step elution, 5 column volumes of elution buffer are usually sufficient. For gradient elution, a shallow gradient (e.g., 20 column volumes or more) can be used to separate proteins with different binding strengths.
After elution in the above steps, wash the medium first with 3 column volumes of Lysis Buffer, then with 5 column volumes of pure water, and finally with 2 column volumes of 20% ethanol. Store the medium at 2–8 °C.
2.5 SDS‑PAGE Analysis
Analyze the purity of the purified product by SDS‑PAGE using the collected fractions (flow‑through, wash, and elution fractions) alongside the original sample.
3. Cleaning‑in‑Place (CIP)
When the resin shows increased backpressure or visible contamination during use, a Cleaning‑in‑Place (CIP) procedure should be performed. The following operations are recommended to remove residual contaminants such as precipitated proteins, hydrophobic proteins, and lipoproteins from the resin.
Removal of strongly hydrophobically bound proteins, lipoproteins, and lipids
Wash with 30% isopropanol for 5–10 column volumes with a contact time of 15–20 min to remove such contaminants. Then wash with 10 column volumes of deionized water. Alternatively, use an acidic or alkaline solution containing detergent for 2 column volumes. For example, 0.1 M acetic acid containing 0.1–0.5% non‑ionic detergent, with a contact time of 1–2 h. After detergent treatment, wash with 5 column volumes of 70% ethanol to thoroughly remove the detergent. Finally, wash with 10 column volumes of deionized water.
Removal of ionically bound proteins
Wash with 1.5 M NaCl solution for 10–15 min. Then wash with 10 column volumes of deionized water. The cleaned column can be rinsed with 2 column volumes of 20% ethanol and stored at 2–8 °C.
4. Resin Regeneration
The cobalt ions on the His‑tag affinity purification resin do not need to be stripped and recharged frequently. When the resin color becomes lighter or the binding capacity noticeably decreases during use, it is necessary to strip the cobalt ions and recharge them—this is resin regeneration. Pack the resin in a suitable chromatography column and follow the procedure below for cobalt stripping and recharging.
1.Wash the resin with 5 column volumes of deionized water.
2.Strip the cobalt ions with 5 column volumes of 100 mM EDTA (pH 8.0).
3.Wash the resin with 10 column volumes of deionized water.
4.Wash with 5 column volumes of 0.5 M NaOH and hold for 10–15 min.
5.Wash the resin with deionized water until the pH is neutral.
6.Recharge cobalt by washing with 3–5 column volumes of 100 mM CoCl₂.
7.Wash with 10 column volumes of deionized water.
After regeneration, the resin can be used immediately. If not used immediately, suspend the resin in an equal volume of 20% ethanol and store at 2–8 °C.
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