For more technical information about PhyTip Affinity Columns, see the Technology Affinity Columns section. PhyNexus introduces IMAC (Immobilized Metal Affinity Chromatography) PhyTip® columns to purify or immobilize functional, recombinant His-tagged protein or antibody quickly at high purity and concentration.
- IMAC affinity columns routinely produce samples of greater than 90% purity
- 60-80% recovery of sample protein.
The image to the left shows an SDS-PAGE gel of His-tagged ubiquitin (His-Ub) enriched from varying levels of background proteins using IMAC PhyTip columns. Purified protein was eluted from the column with 250 mM imidazole. 10 µL of each sample was loaded per lane.
Lane 2: 200 µg/mL of the pure His-Ub
Lanes 3,4: Duplicate enrichments from 200uL PBS spiked with His-Ub to 5 µg/mL
Lanes 5,6: Duplicate enrichments from 200uL PBS, 1% TritonX-100 spiked with His-Ub to 5 µg/mL
Lanes 7,8: Duplicate enrichments from 200uL PBS spiked with His-Ub to 5 ug/mL and BSA to 1 mg/mL
Lanes 9, 10: Duplicate enrichments from 200uL PBS, 1% TritonX-100 spiked with His-Ub to 5 µg/mL and BSA to 1 mg/mL
Conclusion
1. Recover 60-80% of fully functional protein. Pre PhyTip column enrichment concentrations (5 µg/mL) were 40X lower than the pure His-Ub control (200 µg/mL). However, similar recovery of the PhyTip column enriched samples is shown in lanes 3-10 compared to the control in lane 2, demonstrating 60-80% recovery.
2. Obtain greater than 90% purity. The purity is demonstrated by the absence of background proteins.