Background Introduction
This kit provides a rapid, efficient, and convenient protocol for the one-step purification of recombinant proteins tagged with 6×His, 8×His, or 10×His from lysates of Escherichia coli, yeast, insect, and mammalian expression systems. Its core component is Nickel-Iminodiacetic Acid (Ni-IDA) affinity resin. The IDA ligand chelates Ni²⁺ ions in a tridentate manner, leaving sufficient coordination sites for binding to histidine tags. Featuring high binding capacity and cost-effectiveness, this resin is especially suitable for large-scale purification and initial screening experiments.
Product Features
1. Rapid and Efficient: The kit uses IDA-chelated nickel resin with high binding efficiency, which shortens operation time and effectively prevents degradation or denaturation of the target protein during prolonged handling, fully preserving protein activity.
2. Convenient: All necessary reagents for His-tagged protein purification are included, greatly facilitating the purification process.
3. High Binding Capacity: The dynamic binding capacity reaches 35 mg/mL resin.
4. Wide Applicability: The kit supports purification of His-tagged proteins under both native and denaturing conditions. It provides separate lysis buffers, wash buffers, and elution buffers optimized for native and denaturing conditions, satisfactorily meeting diverse experimental requirements.
Instruction For Use
Choose the appropriate buffer system for native or denaturing purification from the reagents provided in the kit.
I. Sample Preparation
A. For Native Buffer System
1. Soluble Protein Expressed in Bacteria or Yeast
(1) Pick a single colony into medium containing the appropriate antibiotic. Induce expression with inducer at the recommended concentration and duration according to the vector instructions.
(2) After expression, transfer the culture to a centrifuge bottle and centrifuge at 7000 rpm (7500 ×g) for 15 min to harvest cells. Resuspend the cell pellet in Native Lysis Buffer at a ratio of 1:10 (w/v). Optionally add PMSF to a final concentration of 1 mM or other protease inhibitors if the protein is prone to degradation, provided they do not interfere with binding to the resin. Lysozyme may be added as needed (not required if the host strain contains pLysS or pLysE).
(3) Fully resuspend the cell pellet. If the cell suspension is concentrated, add 10 μg/mL RNase A and 5 μg/mL DNase I. Mix well, keep on ice, and lyse by sonication in an ice-water bath until the lysate becomes clear.
(4) Transfer the lysate to a centrifuge tube and centrifuge at 10000 rpm (15000 ×g) at 4 °C for 20-30 min. Collect the supernatant, keep on ice, and proceed to purification.
2. Soluble Protein Expressed in Yeast, Insect, or Mammalian Cells
Transfer the cell culture to a centrifuge bottle and centrifuge at 5000 rpm (3800 ×g) for 10 min. Collect the supernatant. If the supernatant contains no EDTA, histidine, or reducing agents, it may be loaded directly. If EDTA, histidine, or reducing agents are present, dialyze against wash buffer before loading. For large volumes, concentrate by ammonium sulfate precipitation and dialyze against wash buffer before loading.
B. For Denaturing Buffer System
1. Inclusion Body Protein Expressed in Bacteria
(1) Pick a single colony into medium containing the appropriate antibiotic. Induce expression with inducer at the recommended concentration and duration according to the vector instructions.
(2) After expression, transfer the culture to a centrifuge bottle and centrifuge at 7000 rpm (7500 ×g) for 15 min to harvest cells. Resuspend the cell pellet in Native Lysis Buffer at a ratio of 1:10 (w/v). Optionally add PMSF to a final concentration of 1 mM or other protease inhibitors if the protein is prone to degradation, provided they do not interfere with binding to the resin. Lysozyme may be added as needed (not required if the host strain contains pLysS or pLysE).
(3) Fully resuspend the cell pellet. If the cell suspension is concentrated, add 10 μg/mL RNase A and 5 μg/mL DNase I. Mix well, keep on ice, and lyse by sonication in an ice-water bath until the lysate becomes clear.
(4) Centrifuge at 10000 rpm (15000 ×g) at 4 °C for 20-30 min. Discard the supernatant and resuspend the pellet in Denaturing Lysis Buffer. Incubate at room temperature for approximately 60 min. Homogenization or sonication may be required to fully dissolve the pellet.
(5) Centrifuge at 12000 rpm for 30 min to remove insoluble debris. Carefully transfer the supernatant to a clean tube without disturbing the pellet, keep on ice, and proceed to purification.
II. Sample Purification
A. Gravity Column Purification
1. Column Packing
(1) Take an empty gravity column, insert the bottom frit, rinse with purified water, and close the bottom outlet.
(2) Mix the Ni-IDA affinity resin thoroughly. Pipette the slurry into the column. The settled resin volume is approximately half of the slurry volume (e.g., 1 mL slurry yields ~0.5 mL resin, with a maximum capacity of ~17.5 mg protein). Open the outlet to drain the storage solution.
(3) Wash the resin with purified water. Drain completely and close the outlet.
(4) Insert the top frit, ensuring no air gap between frit and resin and that the frit is level.
(5) The packed column may be equilibrated immediately with wash buffer. If not used immediately, add storage solution and store at 4 °C.
2. Equilibration
Equilibrate the column with 2-5 CV Wash Buffer (CV = settled resin volume; this definition applies throughout the protocol). Repeat 2-3 times.
3. Sample Loading
Apply the sample to the equilibrated column. Allow the sample to flow slowly to ensure sufficient binding. Collect the flow-through; reloading may increase binding efficiency. Keep samples on ice if the target protein is unstable.
4. Washing
Wash with 5 CV Wash Buffer to remove non-specifically bound proteins. Collect the wash fraction. Imidazole concentration may be adjusted if needed. During the column washing and subsequent elution steps, the protein concentration in each wash and elution fraction can be quickly and simply determined using the Bradford method, allowing you to adjust the number of washing and elution steps accordingly.
5. Elution
Elution buffer contains imidazole as a competitive agent and may be diluted to form step gradients (e.g., 10 mM, 20 mM, 50 mM, 100 mM, 250 mM, 500 mM). Elute with 5 CV Elution Buffer, collect fractions every 1-5 CV, and analyze individually. This ensures complete elution and yields highly pure, concentrated protein.
B. Batch Purification
1. Resin Preparation
Mix the Ni-IDA affinity resin thoroughly. Pipette the desired slurry volume into a centrifuge tube. Centrifuge at 1000 ×g for 2 min and carefully remove the supernatant (low speed to avoid damaging the resin). Alternatively, drain the storage solution in a gravity column.
2. Resin Equilibration
Add 2-5 CV Wash Buffer, mix gently by inversion, centrifuge at 1000 ×g for 2 min, and remove the supernatant. For gravity columns, wash and drain by gravity. Repeat 2–3 times.
3. Binding
Add the prepared sample to the equilibrated resin. Seal the tube or column and mix by rotation. Incubate at room temperature for 30 min or at 2-8 °C for 1 h (longer if needed). Keep at 2-8 °C if the protein is unstable. Centrifuge at 1000 ×g for 2 min and collect the supernatant for analysis.
4. Washing
Wash with 2-5 CV Wash Buffer, mix gently, centrifuge at 1000 ×g for 2 min, and collect the supernatant. For gravity columns, wash and drain by gravity.
5. Elution
Elute using a step imidazole gradient (10 mM, 20 mM, 50 mM, 100 mM, 250 mM, 500 mM). Add 2-5 CV Elution Buffer, mix gently, centrifuge at 1000 ×g for 2 min, and collect the supernatant. For gravity columns, collect eluate by gravity flow.
III. Sample Detection and Resin Handling
1. SDS-PAGE
Analyze samples from the purification process (starting material, flow-through, wash, and elution fractions) by SDS-PAGE to evaluate purification efficiency.
2. Resin Cleaning (In-Place Cleaning)
Cleaning is required to avoid cross-contamination or if backpressure increases due to contamination.
(1) For ionically bound proteins: Wash with 2-3 CV of 2 M NaCl, then flush with ≥3 CV purified water.
(2) For precipitated, hydrophobic, or lipoprotein contaminants: Wash with 2-3 CV of 1 M NaOH for 10-20 min, then flush with water until neutral.
(3) For hydrophobic proteins, lipids, and lipoproteins: Wash with 5-10 CV of 70% ethanol or 30% isopropanol for 15-20 min, then flush with 10 CV water. Alternatively, clean with 2 CV of acidic or alkaline buffer containing detergent (e.g., 0.1-0.5% non-ionic detergent + 0.1 M acetic acid for 1-2 h), then wash with 5-10 CV of 70% ethanol to remove detergent, followed by 10 CV water. After cleaning, the resin may be used immediately or stored in 20% ethanol at 4 °C.
3. Resin Regeneration
Stripping and re-charging with Ni²⁺ is not routinely required. Perform regeneration if the resin becomes pale or binding capacity decreases significantly.
(1) Wash with 5 CV deionized water.
(2) Strip Ni²⁺ with 5 CV of 50 mM EDTA, pH 8.0.
(3) Wash with 10 CV deionized water.
(4) Clean with 0.5 M NaOH (5 CV) and incubate 10-15 min.
(5) Wash with deionized water until neutral.
(6) Re-charge with 3-5 CV of 100 mM NiSO₄.
(7) Wash with 10 CV deionized water. After regeneration, the resin may be used immediately or stored in 20% ethanol at 4 °C.
Matters needing attention
1. Do not freeze this product.
2. The Ni-IDA Affinity Resin must be mixed thoroughly by inversion before use to fully resuspend the agarose beads.
3. Reagents provided are suitable for the purification of most His-tagged proteins.
4. During purification, avoid high concentrations of Tris, HEPES, or MOPS. Do not use chelating agents such as EDTA or EGTA, or reducing agents such as glycine, DTT, or DTE. Optimal concentrations: Triton, Tween, NP-40 up to 1%; guanidine hydrochloride up to 6 M; urea up to 8 M. Although Ni-IDA resin tolerates 6 M guanidine·HCl or 8 M urea, binding capacity is reduced under high denaturant conditions.
5. The Ni-IDA resin slurry contains approximately 50% settled solids per mL. The dynamic binding capacity of the settled resin is up to 35 mg/mL, so each mL of slurry binds approximately 10-17.5 mg protein. Actual maximum binding depends on the molecular weight and properties of the tagged protein.
6. For your safety and health, wear a lab coat and disposable gloves during all experiments.
