Cu IDA Affinity Chromatography Medium (His‑tag) is suitable for the purification of histidine‑tagged (6×His‑tagged) proteins from various expression sources, including E. coli, yeast, insect cells, and mammalian cells. The medium is based on 4% agarose gel as the matrix, onto which tridentate iminodiacetic acid (IDA) is chemically coupled, and then copper ions (Cu²⁺) are chelated. However, copper ions are susceptible to attack by small molecules, and can be reduced or chelated, thereby disrupting the structure and losing binding capacity. The binding strength of copper ions is higher than that of nickel ions. When nickel ion affinity is not satisfactory, Cu IDA Affinity Chromatography Medium (His‑tag) can be selected. Specific performance is shown in Table 1.
Aladdin’s Cu IDA Affinity Chromatography Medium (His‑tag) is stored in 1×PBS containing 20% ethanol, with a gel‑to‑preservative solution volume ratio of 1:1. Our product specifications refer to the actual gel volume.
Table 1: Performance specifications of Cu IDA Affinity Chromatography Medium (His‑tag)
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2. Purification Protocol
2.1 Buffer Preparation
You may use the recommended buffers below or prepare your own buffer systems according to your preferences. The basic principle is low‑imidazole loading and high‑imidazole elution. It is recommended to filter all buffers through a 0.22 μm or 0.45 μm membrane before use.
Table 2: Buffer formulations for soluble His‑tagged protein purification
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2.2 Sample Preparation
2.2.1 Proteins expressed in bacteria or yeast (intracellular soluble)
1.Pick a single colony into culture medium and add the appropriate concentration of inducer according to the vector instructions for the required induction time.
2.After expression, centrifuge the culture at 7,000 rpm (7,500×g) for 15 min to collect the cell pellet. Resuspend the pellet in Lysis Buffer at a ratio of 1:10 (w/v) and add PMSF to a final concentration of 1 mM. Add lysozyme (working concentration 0.2–0.4 mg/mL; this may be omitted if the host strain carries pLysS or pLysE). Other protease inhibitors may be added provided they do not interfere with protein binding.
3.Resuspend the cell pellet thoroughly (if the cell suspension is very viscous, RNase A at 10 μg/mL and DNase I at 5 μg/mL may be added). Disrupt the cells on ice by sonication until the suspension becomes clear.
4.Centrifuge the lysate at 10,000 rpm (15,000×g) for 20–30 min at 4 °C. Collect the supernatant and keep it on ice for immediate use or store at –20 °C.
2.2.2 Secreted soluble proteins from yeast, insect, and mammalian cells
1.Centrifuge the cell culture medium at 5,000 rpm (3,800×g) for 10 min and collect the supernatant. If the supernatant contains no EDTA, histidine, reducing agents, etc., it can be loaded directly onto the column. If such substances are present, dialyse against Lysis Buffer before loading.
2.For large volumes of supernatant, concentrate by ammonium sulphate precipitation and then dialyse against Lysis Buffer before loading onto the column.
2.3 Packing of Cu-IDA Affinity Resin (His tag)
1.Place the lower frit into a suitable gravity column, rinse the column and frit with distilled water, and close the bottom outlet.
2.Mix the Cu-IDA Affinity Resin (His tag) suspension thoroughly and pipette the appropriate amount of slurry into the column (the actual gel volume is half of the suspension volume). Open the bottom outlet to drain the preservative solution.
3.Wash the medium with a suitable volume of distilled water and allow it to drain by gravity. Close the bottom outlet.
4.Insert the pre‑rinsed upper frit, ensuring no air gap between the frit and the medium and that it is level.
5.The packed column can be equilibrated immediately. If not used immediately, cover with preservation solution and store at 4–30 °C.
2.4 Purification Procedures
2.4.1 Batch (incubation) purification
1.According to the sample volume, take an appropriate amount of Cu-IDA Affinity Resin (His tag) into a centrifuge tube and centrifuge at 1,000 rpm for 1 min, then aspirate the supernatant. Alternatively, place the medium in a gravity column and allow the preservative solution to drain.
2.Wash the medium with 5 bed volumes of Lysis Buffer, 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 this washing step at least twice.
3.Add the sample, close the 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. Keep the supernatant as flow‑through for SDS‑PAGE analysis.
5.Wash the medium with 5 bed volumes of Wash Buffer, centrifuge at 1,000 rpm for 1 min or filter in a gravity column, and remove the supernatant (be careful not to disturb the medium). Repeat this washing step 3–5 times, and it is recommended to change to a fresh tube in between. The imidazole concentration in the wash buffer can be adjusted if necessary.
6.Elute with 3–5 bed volumes of Elution Buffer. Incubate at room temperature for 10–15 min, then 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 Cu-IDA Affinity Resin (His tag) gravity column with 5 bed volumes of Lysis Buffer, repeating 2–3 times, so that the medium is in the same buffer system as the target protein.
2.Apply the sample to the equilibrated column and 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 reapplied to increase binding efficiency.
3.Wash with 10–15 bed volumes of Wash Buffer to remove non‑specifically bound contaminants. Collect the wash fractions. The imidazole concentration can be adjusted if needed.
4.Elute with 5–10 bed volumes of Elution Buffer and collect fractions in separate tubes (one column volume per tube). Assay each fraction to ensure complete elution of bound target protein while obtaining high purity and concentration.
Post‑elution treatment: After elution, rinse the column with 3 bed volumes of Lysis Buffer, followed by 5 bed volumes of distilled water, and finally 2 bed 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)
Perform CIP when the column back‑pressure becomes excessively high or when visible contamination of the medium occurs.
Removal of strongly hydrophobically bound proteins, lipoproteins, and lipids:
Wash with 5–10 bed volumes of 30% isopropanol, with a contact time of 15–20 min, followed by 10 bed volumes of distilled water. Alternatively, use an acidic or alkaline solution containing a detergent, e.g., 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 bed volumes of 70% ethanol to completely remove the detergent, then rinse with 10 bed volumes of distilled water.
Removal of ionically bound proteins:
Wash with 1.5 M NaCl for 10–15 min, followed by 10 bed volumes of distilled water. After cleaning, the column can be rinsed with 2 bed volumes of 20% ethanol and stored at 2–8 °C.
4. Resin Regeneration
The copper ions on the His‑tag affinity medium do not need to be stripped and re‑charged frequently. Regeneration is required only when the medium colour becomes noticeably lighter or when the binding capacity significantly decreases. To strip and re‑charge copper ions, pack the medium in a suitable chromatography column and follow the procedure below:
1.Wash with 5 bed volumes of distilled water.
2.Strip copper ions with 5 bed volumes of 100 mM EDTA (pH 8.0).
3.Wash with 10 bed volumes of distilled water.
4.Wash with 5 bed volumes of 0.5 M NaOH and allow to stand for 10–15 min.
5.Wash with distilled water until the effluent reaches neutral pH.
6.Re‑charge with 3–5 bed volumes of 100 mM CuSO₄.
7.Wash with 10 bed volumes of distilled water.
After regeneration, the medium can be used immediately. If not used immediately, suspend it in an equal volume of 20% ethanol and store at 2–8 °C.
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