PROFINITYIMACNi-charged树脂，10m

 

Bio-Rad's Profinity IMAC Ni-charged and uncharged resins provide optimal purification of recombinant His-tagged proteins.

• Optimal ligand density for higher purity of target protein
• Compatibility with denaturants, detergents, and reducing agents for purification over an expanded range of conditions
• Stability in pH 1?4 for storage in a variety of solutions
• Profinity polymer bead for purification at fast flow rates
• Easy-to-pack resin for use in medium-pressure, gravity-flow, and spin columns
• Available uncharged or precharged with Ni²⁺

Profinity IMAC resins provide purification of recombinant His-tagged proteins over a wide molecular weight range. The Profinity IMAC bead is a porous 60 � particle derivatized with iminodiacetic acid (IDA), which functions as the chelating ligand. The chemical structure of IDA, when charged with Ni²⁺ or other transition metal ions, allows highly selective binding of recombinant His-tagged proteins over naturally occurring His-containing proteins. The polymeric nature, optimized IDA ligand density, and open pore structure of the Profinity IMAC bead result in superb mechanical strength, high selectivity for target proteins, low nonspecific binding, and the ability to perform purifications at faster flow rates.

Partial structure of Profinity IMAC resin. Illustration of UNOsphere bead with coupled IDA functional liqand, shown charged with Ni²⁺ . Wavy lines indicate available binding sites.

Profinity IMAC resins, based on patented* UNOsphere technology, exhibit excellent flow properties without compromising binding, capacity, recovery, or purity.

Comparison of maximum operating pressure for different IMAC resins. The values shown in the boxes are the estimated maximum operating pressures for each resin. Beyond these points, the column beds are seriously damaged. Resins were converted to a 50% (v/v) slurry in 20 mM sodium phosphate buffer and packed in a 1.1 x 20 cm column to a bed height of 20 cm up to 43 psi (3 bar). Flow rates were then increased stepwise to 200 cm/hr and held for 2 min at each step. The pressure-flow curve for Profinity IMAC became nonlinear only at pressures above 109 psi, the point defined as the intersection of the two tangents on the pressure-flow curve.

Profinity IMAC resins are stable across the full pH range (1?4) and are compatible with reagents traditionally used in small and large volumes. The resins are easy to pack in Bio-Scale medium-pressure, Econo-Column gravity-flow, and Bio-Spin columns.

Purification of a putative aminopeptidase protein using different IMAC resins. An insoluble 32 kD protein obtained from Anabaena sp. strain PCC 7120 (courtesy of Dr Ray Stevens, University of California, Berkeley, CA, USA) was expressed in E. coli and purified under denaturing conditions. E. coli lysate was loaded onto Micro Bio-Spin columns containing individual IMAC resins and purified. The binding buffer was 50 mM potassium phosphate, 300 mM NaCl, 8 M urea (pH 8.0), and the elution buffer was binding buffer plus 250 mM imidazole. To determine purity of the target protein, 3 � of sample eluate from each column was loaded and separated on a Criterion gel, stained with Coomassie Blue, then quantitated using Quantity One software. Lanes 1 and 8, 10 � Precision Plus Protein standards; lane 2, 3 � crude lysate; lane 3, Ni-charged, high-binding-capacity agarose-based resin from supplier A (IDA ligand); lane 4, uncharged agarose-based resin from supplier A (IDA ligand), charged with Ni²⁺ ; lane 5, Profinity IMAC Ni-charged resin; lane 6, Ni²⁺ -charged agarose-based resin from supplier B (NTA ligand); lane 7, Co²⁺ -charged tetradentate agarose from supplier C. The purity obtained for each resin is indicated; arrow highlights result obtained with Profinity IMAC resin.

* US patent 6,423,666.