His-Tag Protein Purification Using Nickel Resin

Materials Needed

Reagents

• Nickel affinity resin (Cat# 700301)
• Ni-A Buffer (Binding/Wash Buffer): 20 mM Tris, 300 mM NaCl, pH 8.0
• Elution buffer: Ni-A Buffer containing 200-500 mM imidazole
• Optional Additives: Imidazole (10-40 mM for binding buffer or 20-100 mM for wash buffer)

Equipment

• Gravity flow column (e.g., chromatography column)
• Tube rotator or stir plate (for batch purification)
• Collection tubes
• UV spectrophotometer or Bradford assay reagents
• Centrifuge or filtration unit for sample clarification

Protocol

1. Resin Preparation
   1. Gently resuspend the nickel resin by inverting the container until the slurry is homogeneous.
   2. Transfer the desired volume of resin to an empty column.
   3. Allow the resin to settle by gravity.
   4. Drain the storage buffer completely without allowing the resin to dry.
   5. Wash the resin with ≥2 column volumes (CV) of Ni-A buffer to remove residual ethanol from storage.
   The resin is now equilibrated and ready for binding.
2. Binding of His-Tagged Protein
   Column Method
   1. Clarify the lysate by centrifugation or filtration.
   2. Apply the lysate or culture supernatant to the equilibrated column.
   3. Maintain a flow rate of ~0.5–1.0 mL/min.
   4. Collect the flow-through for analysis if desired.
   Batch Method
   1. Add equilibrated nickel resin directly to the clarified lysate.
   2. Incubate with gentle mixing (50–70 rpm) using a rotator or stir plate.
   3. Incubate for: 3–5 hours at room temperature, or overnight at 4°C for optimal binding.
   4. Transfer the resin–sample mixture to a chromatography column.
   5. Allow the resin to settle and drain the unbound fraction.
   Optional: Add 10–40 mM imidazole to the lysate prior to binding to reduce nonspecific protein interactions.
3. Washing
   1. Wash the resin with several column volumes of Ni-A buffer.
   2. Continue washing until background protein levels are minimal.
   3. Monitor the wash fractions using UV absorbance (A280) or a Bradford assay.
   4. For higher purity, include 20–100 mM imidazole in the wash buffer to remove weakly bound contaminants.
4. Elution
   1. Elute the His-tagged protein using Ni-A buffer containing 200–500 mM imidazole.
   2. Collect fractions during elution.
   3. Analyze fractions by SDS-PAGE or absorbance to identify those containing the purified protein.
   4. Pool the desired fractions.
   The optimal imidazole concentration for elution should be determined experimentally for each protein.
5. Column Cleaning After Use
   1. Wash the column with 5 column volumes of deionized water.
   2. Clean the resin with 1 M NaOH at 1–2 mL/min for ~20 minutes.
   3. Re-equilibrate with 5–10 column volumes of Ni-A buffer before reuse.
6. Resin Regeneration (Optional)
   To fully regenerate the resin:
   1. 6 M Guanidine-HCl – 2–5 CV (denaturing wash)
   2. 100 mM EDTA – 2–5 CV (remove nickel ions)
   3. Deionized water – 5–10 CV
   4. 0.1 M NiCl2 – 2–5 CV (recharge resin)
   5. Deionized water – 5–10 CV
   6. Ni-A buffer – 5–10 CV
   7. 20% ethanol – 5–10 CV (optional for storage)
   The resin can typically be reused up to ~10 times without significant loss in binding capacity. Store at 4 °C in PBS containing 20% ethanol for long-term storage.
7. Storage
   1. Store resin at 4°C.
   2. Short-term: Ni-A buffer (1–2 months).
   3. Long-term: Ni-A buffer with 20% ethanol.

Tips and Tricks

• Clarify lysates thoroughly - Residual debris can clog columns and reduce binding efficiency. Centrifuge and filter lysates before loading.
• Optimize imidazole concentrations - Small changes in imidazole concentration during washing can dramatically improve purity.
• Use protease inhibitors – Proteolysis during purification can damage the His-tag or degrade your target protein.
• Maintain appropriate pH - His-tag binding is strongest at pH 7.5–8.5.
• Keep proteins cold if unstable - Perform purification at 4°C if your protein is sensitive to degradation or aggregation.
• Analyze fractions during optimization - Running SDS-PAGE on flow-through, wash, and elution fractions helps identify losses.

Troubleshooting Guide

Problem	Possible Cause	Solution
Low protein yield	His-tag inaccessible or degraded	Confirm tag sequence and position; test N- vs C-terminal tags
Target protein in flow-through	Insufficient binding time	Increase incubation time or reduce flow rate
High background proteins	Nonspecific binding	Increase wash imidazole concentration
Protein does not elute	Imidazole concentration too low	Increase to 400–500 mM
Resin turns brown/black	Metal contamination or oxidation	Regenerate resin with EDTA and recharge with NiCl2
Column clogging	Lysate not clarified	Centrifuge and filter samples before loading