Ni-NTA Agarose (100 ml)

A nickel-charged affinity chromatography resin from QIAGEN, this 100 ml bottle of settled gel supports the purification of recombinant proteins carrying a polyhistidine tag via immobilized nickel ions bound to nitrilotriacetic acid (NTA) ligands. Ni-NTA Agarose combines these NTA groups with cross-linked 6% agarose beads, with the immobilized Ni²⁺ ions coordinating the imidazole side chains of consecutive histidine residues. This chelation captures 6xHis-tagged proteins from crude bacterial, yeast, or mammalian lysates under both native and denaturing conditions.

The settled gel is used in gravity-flow columns, batch binding in conical tubes, or packed into FPLC and HPLC systems. Each milliliter of resin routinely binds up to 40 mg of His-tagged protein, while the bead size range of 45-165 µm balances flow rate with surface area for efficient capture and elution. Working pH spans 2-14, allowing washes with 8 M urea, 6 M guanidine-HCl, or imidazole gradients up to 20 mM without resin degradation.

For elution, 250-500 mM imidazole at pH 7-8 is standard, while lower pH buffers (pH 4.5-5.3) can displace bound protein when imidazole must be avoided. The resin tolerates common additives such as 2 mM DTT, 1 mM EDTA, and up to 30% isopropanol or 20% ethanol for storage and sanitization. Store refrigerated at 4°C in 20% ethanol to prevent microbial growth and maintain nickel loading.

Typical workflows include purification of His-tagged kinases, transcription factors, viral antigens, and antibody fragments for crystallography, enzymatic assays, and immunization studies. The same chemistry also enables pull-down assays, on-bead enzymatic reactions, and immobilization of metalloproteins for activity studies.