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| Product Name | Ni-NTA Magnetic Beads, 2 µm, for His-Tag Protein Purification, Low Nickel Leaching |
| Catalog No. | MAGBEA-0031 |
| Description | Nickel-nitrilotriacetic acid (Ni-NTA) magnetic beads for rapid single-step affinity purification of recombinant proteins containing polyhistidine (6×His, 10×His) tags from E. coli, yeast, insect, and mammalian cell lysates under native or denaturing conditions. NTA is a tetradentate chelating ligand that occupies four of the six nickel coordination sites, leaving two sites for histidine binding, providing stronger metal coordination and lower nickel leaching compared to tridentate iminodiacetic acid (IDA) chemistry. The low nickel leaching formulation reduces protein oxidation and nickel toxicity artifacts in downstream functional and cell-based assays, and the beads are compatible with on-column refolding of inclusion body proteins solubilized with urea or guanidine. |
| Intended Use | Affinity purification of 6×His, 10×His, and other polyhistidine-tagged recombinant proteins from bacterial, yeast, insect, and mammalian expression systems for structural biology, enzymology, antibody production, and functional studies. |
| Principle / Technology | Ni2+ ions chelated by NTA ligand on bead surface coordinate with imidazole side chains of histidine residues in polyhistidine tag; binding affinity increases with histidine tag length (6×His Kd ~1-10 µM; 10×His Kd ~0.1-1 µM); competitive elution with imidazole (150-500 mM); low nickel leaching compared to IDA-based resins. |
| Detection Method | Equilibrate beads in binding buffer; add to clarified lysate; incubate 30-60 min at 2-8 °C or 15-30 min at RT with rotation; magnetically separate; wash 3× with wash buffer (containing 10-50 mM imidazole); elute with elution buffer containing 150-500 mM imidazole; dialyze or desalt eluate to remove imidazole if needed. |
| Sample Type | Clarified E. coli lysates (soluble fraction or urea/guanidine-solubilized inclusion bodies); yeast and insect cell lysates; mammalian cell lysates; in vitro translation reactions. |
| Performance Range / Specifications | Binding capacity: 20-50 mg 6×His-tagged protein per mL settled beads (protein-dependent, typical for 30-50 kDa protein); typical purity >90% from E. coli lysate in single step. |
| Sensitivity / LOD | Binding of His-tagged proteins at concentrations as low as 0.1 µg/mL in lysate; detectable recovery from 50 mL bacterial culture expressing at moderate levels (5-10 mg/L). |
| Specificity | Selectivity for polyhistidine tag based on metal chelate affinity; minimal binding of endogenous E. coli histidine-rich proteins at 10-20 mM imidazole in wash buffer; increasing imidazole stringency improves purity at cost of yield. |
| Reaction Conditions / Protocol | Binding: 30-60 min at 2-8 °C; wash with 10-50 mM imidazole; elute with 150-500 mM imidazole; compatible with native, urea (up to 8 M), and guanidine (up to 6 M) conditions. |
| Components / Formulation | Ni-NTA magnetic bead suspension (50% slurry in 20% ethanol), 10× Binding Buffer (500 mM NaH2PO4, 3 M NaCl, pH 8.0), 5 M imidazole solution, detailed protocol for native and denaturing purification. |
| Storage Conditions | Store beads at 2-8 °C in 20% ethanol; do not freeze; imidazole solution at 2-8 °C. |
| Shelf Life | 12 months from date of manufacture. |
| Package Specifications | 1 mL settled beads (2 mL 50% slurry), 5 mL, 25 mL, 100 mL. |
| Product Form | Dark brown/black magnetic bead suspension; slurry formula in 20% ethanol. |
| Quality Control | Each lot tested for binding capacity using 6×His-GFP standard (≥30 mg/mL beads), nickel leaching by ICP-MS (<5 ppm Ni in eluate after 24-hour incubation), and purity of single-step purification from E. coli lysate (>85% by SDS-PAGE). |
| Key Features | Ni-NTA chemistry with low nickel leaching; 2 µm uniform magnetic beads; rapid purification (30-60 min); compatible with native and denaturing conditions; on-column refolding possible; suitable for automated liquid handling and high-throughput. |
| Purity | No free Ni2+ detected above 5 ppm; bead suspension sterile; endotoxin <0.1 EU/mL (endotoxin-free option available). |
| Concentration | 50% slurry in 20% ethanol; binding capacity 20-50 mg protein/mL settled beads. |
| Activity / Unit Definition | Ni-NTA binding: Kd ~0.1-10 µM for polyhistidine tag; imidazole elution IC50 ~150 mM for 6×His. |
| Molecular Weight | Not applicable; Ni-NTA-chelate complex molecular weight ~350 Da. |
| Source / Origin | Magnetic iron oxide core with silica or polymer matrix; NTA ligand and Ni2+ loading under controlled conditions. |
| pH Range / Optimal pH | Binding optimal at pH 7.5-8.0; compatible pH range 6.0-8.5; Ni2+ leaching increases below pH 6.0. |
| Shipping Conditions | Cold pack (2-8 °C); do not freeze. |
| Expiration Date / Stability | 12 months at 2-8 °C; beads must be stored in 20% ethanol or equivalent bacteriostatic agent; do not freeze — freezing cracks magnetic bead matrix. |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic applications. |
| Compatibility | Compatible with Tris, phosphate, and HEPES buffers. Avoid EDTA and EGTA (>1 mM) as they strip Ni2+ from NTA. Avoid DTT >10 mM (reduces Ni2+). Compatible with 0.1% nonionic detergents. For denaturing purification: compatible with 8 M urea or 6 M guanidine-HCl; reduce imidazole in binding buffer to 5-10 mM. |
| Recommended Buffer System | Phosphate or Tris-based buffer with 300-500 mM NaCl and 5-50 mM imidazole, pH 7.5-8.0. |
| Application Notes / Precautions | Pre-equilibrate beads in binding buffer (washing 3× with 5 volumes of buffer). Include 5-20 mM imidazole in binding/wash buffer to reduce nonspecific binding of host proteins. For E. coli lysates, add PMSF and lysozyme; sonicate on ice; centrifuge at 15,000 × g for 20 min to clarify. Nickel leaching is minimal with NTA chemistry but for nickel-sensitive applications, strip and recharge beads with fresh NiSO4 before each use. Beads can be stripped with 0.5 M EDTA pH 8.0 and recharged with 0.1 M NiSO4 for regeneration (up to 5-10 cycles). |
| Batch-to-Batch Consistency | Binding capacity within ±20% of reference lot; nickel leaching <5 ppm; bead size uniformity (PDI <0.1). |
For research use only, not for clinical use.
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