Ni-NTA Fast Start Kit

The Ni-NTA Fast Start Kit provides everything required for fast, efficient purification of His-tagged proteins from cleared E. coli lysates. Buffers supplied in the kit enable proteins to be purified either under native or denaturing conditions (see figure High purity proteins under native or denaturing conditions) Simple, straightforward protocols and ready-to-use reagents make the Ni-NTA Fast Start Kit an ideal starting point for scientists looking to expand their research into the field of protein expression, but who are unfamiliar with protein purification procedures.

The Ni-NTA Fast Start Kit is based on the remarkable selectivity of patented Ni-NTA (nickel-nitrilotriacetic acid) resin for proteins containing an affinity tag of six or more histidine residues (consecutive or alternating) — the His tag. This technology allows one-step purification of almost any His-tagged protein under native or denaturing conditions. NTA, which has four chelation sites for nickel ions, binds nickel more tightly than metal-chelating purification systems that only have three sites available for interaction with metal ions. The extra chelation site prevents nickel-ion leaching and results in protein preparations with higher purity than those obtained using other metal-chelating purification systems. The His tag also forms the epitope recognized by the Penta·His Antibody supplied with the kit.

Cells are lysed in the supplied lysis buffer and centrifuged to obtain a cleared lysate. This lysate is applied to a Fast Start Column, where His-tagged proteins bind with high affinity and untagged proteins pass through. After washing, purified proteins are eluted using buffers containing imidazole (native conditions) or of low pH (denaturing conditions) (see figure Highly pure 6xHis-tagged protein). The purification procedure and protein yield can be followed by SDS-PAGE, western blotting and immunodetection using the supplied Penta·His Antibody.

The Ni-NTA Fast Start Kit provides reliable, one-step purification of 6xHis-tagged proteins suitable for any application, including:

• Functional studies
• Immunization to produce antibodies
• Assays involving protein–protein and protein–DNA interactions
• Structural investigations