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| Product Name | Ni-NTA Magnetic Beads (His-tag Protein Purification) |
| Catalog No. | MAGBEA-0014 |
| Description | Nickel-nitrilotriacetic acid (Ni-NTA) functionalized superparamagnetic beads for the high-affinity purification of polyhistidine-tagged recombinant proteins. The tetradentate NTA chelating ligand coordinates Ni²+ ions with high stability, presenting two free coordination sites for hexahistidine tag binding. The robust chelation chemistry minimizes nickel leaching under binding and wash conditions while supporting efficient imidazole-mediated elution under native or denaturing conditions. |
| Intended Use | Purification of His6-tagged recombinant proteins from bacterial, yeast, insect, and mammalian expression systems under native or denaturing conditions for biochemical characterization, structural biology, and functional protein studies. |
| Principle / Technology | NTA ligands are covalently attached to the bead surface via a stable spacer arm. Ni²+ ions are coordinated by the NTA chelator with a binding constant of ~10^6 M^-1, providing four coordination sites to the metal while leaving two coordination positions available for interaction with histidine residues. Polyhistidine tags (typically His6) bind through coordination of adjacent histidine imidazole side chains to the immobilized nickel. Proteins are eluted by competitive displacement with imidazole under mild conditions. |
| Detection Method | BCA assay for protein concentration; SDS-PAGE for purity; Western blot with anti-His antibody; DLS for bead size; nickel loading quantitation by ICP-OES; imidazole gradient elution analysis; enzymatic activity assay of purified proteins |
| Sample Type | His-tagged proteins (His6, His10, His12) from E. coli, yeast, baculovirus/insect, and mammalian cell lysates; compatible with native and denaturing (8 M urea, 6 M guanidine HCl) conditions; whole cell lysates clarified by centrifugation; periplasmic extracts |
| Performance Range / Specifications | Bead diameter: 1-2 µm; nickel loading: 10-25 µmol Ni²+/mL settled beads; protein binding capacity: 20-50 mg His6-GFP/mL settled beads; binding constant: ~10^6 M^-1 for His6 tag; nickel leaching: <1% under standard conditions; EDTA tolerance: avoid during binding; imidazole elution: 50-500 mM gradient; magnetic separation: <10 seconds |
| Sensitivity / LOD | Protein detection from lysates with <1 µg/mL His-tagged target; purification from low-expression clones yielding <100 µg/L culture; imidazole elution at 50 mM removes weakly bound contaminants, 250-500 mM elutes target |
| Specificity | Specific binding to accessible polyhistidine sequences; minimal binding of endogenous E. coli histidine-rich proteins with appropriate imidazole wash (10-20 mM); no binding of untagged proteins; NTA-Ni²+ has higher specificity than IDA-based resins; no binding of non-histidine metal-binding proteins under recommended conditions |
| Reaction Conditions / Protocol | Charge beads with 100 mM NiSO4 if supplied uncharged (or use pre-charged beads directly); equilibrate with binding buffer (50 mM Tris pH 8.0, 300 mM NaCl, 10-20 mM imidazole); add clarified lysate; incubate 30-60 minutes at 4°C with rotation; magnetically separate; wash with binding buffer containing 20-50 mM imidazole; elute with binding buffer containing 250-500 mM imidazole; incubate 5-10 minutes per elution; total protocol: 1-2 hours; beads can be stripped with EDTA and recharged 5-10 times |
| Components / Formulation | NTA-functionalized superparamagnetic beads, 25-50% slurry in 20% ethanol; pre-charged with Ni²+ or supplied without metal for user charging; NiSO4 charging solution provided with uncharged format; binding, wash, and elution buffers not included |
| Storage Conditions | 2-8°C in 20% ethanol; do not freeze; protect from light; store upright; for pre-charged beads, nickel may slowly leach over time—recharge every 3-6 months; minimize exposure to chelating agents during storage |
| Shelf Life | 24 months at 2-8°C in 20% ethanol; nickel binding capacity stable at 24 months; NTA ligand stability verified; pre-charged beads: nickel content stable >90% at 12 months; recharge before use for maximum capacity |
| Package Specifications | 1 mL, 5 mL, 10 mL, 25 mL settled bead volume; pre-charged and uncharged formats; bulk packaging for manufacturing; trial packs available |
| Product Form | Light green (pre-charged Ni²+) or white/off-white (uncharged) bead suspension in 20% ethanol; homogeneous after mixing; nickel-charged beads have characteristic pale green color; color intensity proportional to nickel loading |
| Quality Control | Per lot: nickel loading (ICP-OES), protein binding capacity (His6-GFP standard), nickel leaching (EDTA titration), non-specific protein binding (E. coli lysate), binding capacity after 5 regeneration cycles, endotoxin, sterility, particle size |
| Key Features | High nickel binding capacity with low metal leaching; NTA tetradentate chelator for stable nickel coordination; compatible with native and denaturing conditions; reusable with strip-and-recharge regeneration; EDTA-compatible stripping; imidazole elution preserves protein activity |
| Purity | Ni²+ loading: 10-25 µmol/mL; endotoxin <0.1 EU/mg; nickel leaching <1%; no heavy metal contamination (Co, Zn, Cu <5 ppm) |
| Concentration | 25-50% slurry; nickel 10-25 µmol/mL settled beads; protein binding 20-50 mg/mL settled beads; exact values per lot |
| Activity / Unit Definition | His6-GFP binding: 20-50 mg/mL; Kd ~10^-6 M; imidazole elution >90% at 500 mM; recharging capacity >90% after 5-10 cycles |
| Molecular Weight | NTA ligand: ~268 g/mol; Ni²+: 58.7 g/mol; His6 tag: ~841 Da; bead molecular weight not defined |
| Source / Origin | Synthetic NTA ligand chemically coupled to polymer-magnetite beads; nickel sulfate hexahydrate (NiSO4·6H2O) is analytical grade; beads are fully synthetic; no animal or biological source materials; metal content traceable |
| pH Range / Optimal pH | Binding: pH 7.0-8.5 (optimal 7.5-8.0 for His6); imidazole elution: pH 7.0-8.5; nickel leaching increases below pH 5.5; Ni²+ precipitation above pH 9 in phosphate buffers; bead integrity: pH 2-13 |
| Shipping Conditions | Ambient temperature in 20% ethanol; no special handling; non-hazardous for transport; standard courier |
| Expiration Date / Stability | 24 months in 20% ethanol; NTA ligand stability confirmed; nickel loading stable; real-time monitoring at 0, 12, 24 months; recharge after 6 months for optimal performance with pre-charged beads |
| Regulatory / Compliance | For research use only; not for therapeutic protein production; nickel salt hazard in charging solution; ISO 9001 manufacturing; SDS available; REACH compliant |
| Compatibility | Native lysis buffers: Tris, phosphate, HEPES (50-100 mM, pH 7.5-8.0, 300 mM NaCl); denaturing: 8 M urea or 6 M guanidine HCl in Tris or phosphate pH 7.5-8.0; avoid EDTA, EGTA during binding; β-mercaptoethanol up to 20 mM acceptable; DTT up to 10 mM; imidazole up to 20 mM in binding buffer reduces background |
| Recommended Buffer System | Binding: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10-20 mM imidazole; wash: binding buffer + 20-50 mM imidazole; elution: binding buffer + 250-500 mM imidazole; stripping: 50 mM EDTA pH 8.0; recharging: 100 mM NiSO4; storage: 20% ethanol |
| Application Notes / Precautions | Include 10-20 mM imidazole in binding buffer to reduce non-specific binding; for low-expression targets, extend incubation to 2 hours; imidazole in eluate can be removed by dialysis or desalting; EDTA stripping followed by recharging restores full capacity; avoid phosphate buffers with high nickel concentrations (precipitate formation); for structural biology, use highest purity imidazole available |
| Batch-to-Batch Consistency | Nickel loading CV <12%; protein binding CV <10%; nickel leaching <1% all lots; binding capacity maintained through 5 regeneration cycles; each lot validated with His6-GFP standard |
For research use only, not for clinical use.
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