PROTEINDEX™ HiBond™ Ni-NTA Agarose 6FF

PROTEINDEX™ HiBond™ Ni-NTA Agarose 6FF has been optimized for automated purification applications. It has been widely used in scale-up and preparative purification of histidine-tagged proteins that are expressed in various hosts, such as E.coli, yeast, insect cells, and mammalian cells.         The base matrix of PROTEINDEX™ HiBond™ Ni-NTA Agarose 6FF is highly crosslinked 6% agarose, offering excellent chemical-, and mechanical stability for automated applications. The matrix support is functionalized with the chelate ligand Nitilotriacetic Acid (NTA) in complex with the nickel ions (Ni2+) in a very stable octahedral structure. The structure of Ni-NTA complex is compatible solutions that contain certain concentration of reducing agents, denaturing agents, detergents and other additives.         The NTA ligand coordinates the Ni2+ with four valencies (tetradentate, coordination number 4), and two valencies are available for interaction with imidazole rings of histidine residues. The number of available valencies seems to provide the best combination of binding effectiveness and chemical stability for binding with the His tag. The Ni-NTA ion-chelate complex on our ProteIndex™ HiBond™ Ni-NTA resins has been delicately optimized to ensure the most effective capture for His tag purification of His-tagged proteins.          Product Highlights Mechanical stable, designed for scaling up His tag purification 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 Automated or syringe operations; and manual batch or gravity flow Routine His tag protein purification for structural biology and functional assays Scaling up and preparative applications   Tech Notes One-step purification using ProteIndex™ HiBond™ Ni-NTA Agarose 6 Fast Flow resins Figure 1. A 15 kDa protein was expressed in E.coli and purified on an automated system using ProteIndex™ HiBond™ Ni-NTA Agarose 6 Fast Flow resin under native conditions. 50 mL E.coli lysate in 50 mM NaH2PO4, 0.3 M NaCl, 10 mM imidazole was loaded on the column packed with 0.5 mL resins at the speed of 1 mL/min. The column was then washed with 100 mL 50 mM NaH2PO4, 0.3 M NaCl, 20 mM imidazole at the speed of 1~2 mL/min. The target protein was eluted with 50 mM NaH2PO4, 0.3 M NaCl, 250 mM imidazole. Proteins were visualized by coomassie blue staining after separation on SDS-PAGE. L: raw lysate; F: unbound flow-through; E1 and E2: eluate fractions.   Figure 2. A 25 kDa protein was expressed in E.coli and purified on an automated system using a one-step automated protocol with ProteIndex™ HiBond™ Ni-NTA Agarose 6 Fast Flow resin under native conditions. 50 mL E.coli lysate in 50 mM NaH2PO4, 0.3 M NaCl, 10 mM imidazole was loaded on the column packed with 0.5 mL resins at the speed of 1 mL/min. The column was then washed with 100 mL 50 mM NaH2PO4, 0.3 M NaCl, 20 mM imidazole at the speed of 1~2 mL/min. The target protein was eluted with 50 mM NaH2PO4, 0.3 M NaCl, 250 mM imidazole. A fraction of eluate was analyzed on a SDS-PAGE. Shown were coomassie blue stains of the target proteins purified from columns of Marvelgent’s ProteIndex Ni-NTA 6FF (M) and Ni-Sepharose (G) resins. Product Details Ligand Nickel Binding capacity > 40 mg 6xHis-tagged protein/mL medium Matrix 6% highly cross-linked agarose supplied as 50% slurry Particle size 45 μm - 165 μm Maximum pressure 0.3 MPa (3 bar) Chemical compatibility Reducing agents 5 mM DTE 0.5-1 mM 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.