PrepEase Histidine-tagged High Yield Purification Resin
Dry, silica-based resin, precharged with Ni2+ ions
Binding Capacity up to 20 mg protein per gm of resin
PrepEase® Histidine-tagged High Yield Purification Resin is a dry, silica-based resin, precharged with Ni2+ ions. The resin matrix, IDA (Iminodiacetic Acid), is a tridentate metal chelator which enables strong and efficient binding of target proteins. The PrepEase Ni-IDA Resin has multiple binding sites to Histidine-tagged protein, resulting in higher yields.
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Customer Data 1: Capacity of different Ni2+ affinity chromatography resins. 2.5 ml of solubilized membrane vesicles of L. lactis NZ9000 harboring ~300 μg recombinant (10-His-tagged) MhsTBh per ml (starting material, SM) were incubated with different resins at about 50% their listed binding capacity for 30 min at 4°C in the presence or absence of 20 mM imidazole. The protein/resin mix was loaded onto a chromatography column and the unbound fraction of each sample was collected by gravity flow. The amount of unbound recombinant MhsTBh in the flow-through was detected by immunoblotting after subjecting 2 μl of the sample to 11% SDS-PAGE analysis. The highest binding capacity was achieved with USB PrepEase High Yield resin in the presence (U1) or absence (U2) of imidazole, whereas Ni2+-Fastflow (F1, F2) and Ni2+-Superflow (S1, S2) showed lower binding efficiency for the target protein under the same experimental conditions. Data kindly provided by Matthias Quick, Ph.D., Center for Molecular Recognition, Columbia University. |
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| Customer Data 2: Purification of MhsTBh by immobilized metal chelate affinity chromatography. Membrane vesicles of L. lactis NZ9000 harboring recombinant MhsTBh were solubilized with 2.5% n-decyl-β-D-maltopyranoside for 1 hour at 4°C. The solubilized protein fraction (starting material, SM) was separated from the insoluble material (pellet, P) by centrifugation at 307,000 x g for 30 min at 4°C and used for batch binding with USB PrepEase His-Tag High Yield resin (1 g resin per 10 mg target protein) for 30 min at 4°C. The protein/resin mix was loaded onto a chromatography column, the flow-through was collected (F), and the column was washed with 50 mM imidazole-containing chromatography buffer (not shown). MhsTBh was eluted with 250 mM imidazole (E). 10 μg of total protein of each sample were subjected to 11% SD-PAGE followed by Coomassie staining of the gel and Western blotting. Data kindly provided by Matthias Quick, Ph.D., Center for Molecular Recognition, Columbia University. |
