PROTEINDEX™ HiBond™ Ni-NTA Agarose

PROTEINDEX™ HiBond™ Ni-NTA Agarose utilizes 4% agarose as the matrix support, and is functionalized with the chelate ligand Nitilotriacetic Acid (NTA), which is charged with nickel ions (Ni2+) to form a very stable octahedral structure with the nickel ion in the center. The structure of Ni-NTA complex is compatible with solutions that contain certain concentration of reducing agents, denaturing agents, detergents and other additives. PROTEINDEX™ HiBond™ Ni-NTA Agarose can be used to purify 6xHis-tagged proteins expressed in various hosts, such as E.coli., yeast, insect cells and mammalian cells.   Product Highlights Suited for routine purification of histidine-tagged proteins High yield (typically > 40 mg protein /mL resins) Low leakage of nickel ions. Can be re-used for up to 8 preps* *Note: No significant decrease in yield after 8 repetitive uses of resin when recommended buffers are used. Refer to Product Details for compatible reagents.   Applications Manual batch or gravity flow purification Routine His tag protein purification for structural biology and functional assays Protein-protein and protein-nucleic acid interaction studies   Tech Notes One-step purification using ProteIndex™ HiBond™ Ni-NTA Agarose 6 Fast Flow resins Figure 1. E.coli lysate containing a 50 kDa target protein was directly loaded on HiBond™ Ni-NTA Agarose 6 FF column. The column was then washed sequentially with 0 mM, 5 mM, and 20 mM imidazole solution before elution with 300 mM imidazole. The target protein was analyzed by SDS-PAGE. CL: raw lysate; FT: unbound flow-through; W1, W2 and W3: washes; E1 and E2: eluate fractions. Product Details Ligand Nickel Binding capacity >40 mg 6xHis-tagged protein/mL medium Matrix 4% agarose supplied as 50% slurry Particle size 45 μm - 165 μm Maximum pressure 0.1 MPa (1 bar) Recommended flow rate Gravity flow, <75 cm/hour Chemical compatibility Reducing agents 5 mM DTE 0.5-1mM DTT 20 mM β-mercaptoethanol 5 mM TCEP 10 mM reduced glutathione (GSH) Denaturing agents 8 M urea, 6 M Gua-HCl (Tested for one week at 40°C) Detergents 2% Triton™ X-100 (nonionic) 2% Tween™ 20 (nonionic) 2% NP-40 (nonionic) 2% cholate (anionic) 1% CHAPS (zwitterionic) Other additives 500 mM imidazole 20% ethanol 50% glycerol 100 mM Na2SO4 1.5 M NaCl 1 mM EDTA 60 mM citrate Commonly used buffer 50 mM sodium phosphate, pH 7.4 100 mM Tris-HCl, pH 7.4 100 mM Tris-acetate, pH 7.4 100 mM HEPES, pH 7.4 100 mM MOPS, pH 7.4 100 mM sodium acetate, pH 4 Storage temperature 2 - 8°C Storage buffer 1x PBS containing 20% ethanol   Product is shipped at ambient temperature. Upon receipt store at 2 - 8°C. Do not Freeze.