Ni-NTA Magnetic Agarose Beads

Ni-NTA Magnetic Agarose Beads are magnetic particles coated with Ni-NTA Agarose affinity purification matrix. They are used for immobilizing and purifying recombinant proteins carrying a His tag. Histidine residues in the His tag bind to the vacant positions in the coordination sphere of the immobilized nickel ions with high specificity and affinity. Once proteins are bound, the beads can be precipitated using a magnet, washed, and proteins eluted in small volumes of buffer under native or denaturing conditions.

The QIAexpress Ni-NTA Protein Purification System, including the Ni-NTA Magnetic Agarose Beads (see figure  Micrograph of Ni-NTA Magnetic Agarose Beads) 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 — the His tag. This technology allows one-step purification of almost any His-tagged protein from any expression system under native or denaturing conditions (see figure  Protein purification with the Ni-NTA protein purification system). 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 a greater binding capacity and protein preparations with higher purity than those obtained using other metal-chelating purification systems. The QIAexpress system can be used to purify His-tagged proteins from any expression system including baculovirus, mammalian cells, yeast, and bacteria.

The purification of His-tagged proteins consists of 4 stages: cell lysis, binding, washing, and elution (see figure  Micro-scale protein purification). Purification of recombinant proteins using the QIAexpress system does not depend on the 3-dimensional structure of the protein or His tag. This allows one-step protein purification under either native or denaturing conditions, from dilute solutions and crude lysates. Strong denaturants and detergents can be used for efficient solubilization and purification of receptors, membrane proteins, and proteins that form inclusion bodies. Reagents that allow efficient removal of nonspecifically binding contaminants can be included in wash buffers (see table). Purified proteins are eluted under mild conditions by adding 100–250 mM imidazole as competitor or by a reduction in pH.

The QIAexpress Ni-NTA Protein Purification System, including the Ni-NTA Magnetic Agarose Beads, provides reliable, one-step purification of proteins suitable for any application, including:

• Structural and functional investigations
• Crystallization for determination of three-dimensional structure
• Assays involving protein–protein and protein–DNA interactions
• Immunization to produce antibodies