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Superior performance of HisPur Ni-NTA Magnetic Beads when compared to magnetic beads from other suppliers
Bacterial lysate (100μg total protein) containing over-expressed 6xHis-GFP (Panel A) or over-expressed 6xHis-β Galactosidase (Panel B) was applied to 0.5mg of HisPur Ni-NTA Magnetic Beads, Mag Sepharose™ (GE Healthcare), PureProteome™ (Millipore), and Magnetic Agarose (Qiagen) Ni-NTA bead products. All samples were run in duplicate, and the beads were processed using the Thermo Scientific protocol with buffers recommended by the manufacturers. For the HisPur Ni-NTA beads the amount of imidazole in the Equilibration, Wash and Elution Buffers was 30mM, 50mM and 250mM, respectively. All three buffers contained 100mM sodium phosphate and 600mM sodium chloride. Binding was performed with all samples for 30 minutes. The beads were collected on a magnetic stand and the flow throughs were saved for analysis. The beads were then washed twice and bound protein was eluted for 2 x 15 minutes with Elution Buffer. The eluates were combined, resolved on an SDS-PAGE gel and stained with Thermo Scientific Imperial Stain. Purity analyses were performed on a Thermo Scientific myECL Imager with myImageAnalysis Software. The % purity was determined by measuring the ratio of the background-corrected 6xHis-tagged protein band of interest to the sum of all bands and multiplying by 100%.Comparable yields and comparable % purity were observed for 6xHis-GFP with HisPur Ni-NTA and Qiagen Ni-NTA agarose magnetic beads. HisPur Ni-NTA beads provided higher yield and purity than Qiagen Ni-NTA agarose magnetic beads for purification of 6xHis-β galactosidase. Millipore and GE Ni-NTA magnetic beads gave lower purity and lower yield than Thermo Scientific beads in both purifications.
