Ni-IMAC Columns

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
    • Obtain greater than 90% purity. The purity is demonstrated by the absence of background proteins.
  • 60-80% recovery of sample protein.
    • 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.
Description

PhyTip columns are unique capture, purification and enrichment tools from PhyNexus designed for micro volume protein sample preparation. PhyTip Columns are available for a variety of liquid handling platforms and contain specific affinity resins for application specific requirements. Samples for purification and enrichment must be clear and free from particulate matter. It is highly recommended to centrifuge samples and use the clear supernatant only, prior to use with PhyTip columns.

200+ PhyTip columns with IMAC resin:

For a 200 µL sample with 5 µg His-Tagged Fab containing 1 mg BSA processed using the conditions shown below, greater than 50% of the original HisTagged Fab mass is recovered in the final sample volume. In addition, this recovered His-Tagged is purified to over >95% purity as determined by SDSPAGE with Coomassie detection.

Capture: 200 µL sample captured by passing through the resin bed for two cycles at a flow rate of 250 uL per minute.

Purify: 200 µL of PhyNexus IMAC Wash Buffer I, passed over the resin bed for two cycles at a flow rate of 500 µL/min followed by a second wash with same buffer, passed over the resin bed for two cycles at a flow rate of 500 µL/min.

Enrich: elute the protein into solution with 10µL of IMAC Elution Buffer, passed over the resin bed for five cycles at a flow rate of 500 µL/min.

1000+ PhyTip columns with IMAC resin:

For a 500 µL sample with 5 µg His-Tagged Fab containing 1 mg BSA, processed using the conditions shown below, greater than 50% of the original HisTagged Fab mass is recovered in the final sample volume. In addition, this recovered His-Tagged Fab is purified to over >95% purity as determined by SDSPAGE with Coomassie detection.

Capture: 500 µL sample captured by passing through the resin bed for two cycles at a flow rate of 250 uL per minute.

Purify: 1000 µL of PhyNexus IMAC Wash Buffer I, passed over the resin bed for two cycles at a flow rate 500 µL/min followed by a second wash with the same buffer, passed over the resin bed for two cycles at a flow rate 500 µL/min.

Enrich: elute the protein into solution with 15µL of PhyNexus IMAC Elution Buffer, passed over the resin bed for five cycles at a flow rate of 500 µL/min.

Data

Shipping and Storage

Each pack of PhyTip columns has been manufactured and QC’d to the highest standards and shipped in retainer boxes that maintain the integrity of the specific affinity resin within each PhyTip column. This product is shipped at ambient temperatures, but on receipt should be stored in a standard laboratory refrigerator between 4 and 8◦C.

  • Do NOT freeze or store frozen.
  • When not in use, keep the lid of the box closed and sealed, store in the refrigerator.
  • Do not allow affinity resin to dry out by extended storage in a dry environment.

IMAC PhyTip columns are stored in Glycerol when shipped from PhyNexus.

IMAC PhyTip Columns

IMAC PhyTip columns have been optimized for use with specific PhyNexus reagents and instrument flow rates/volumes as shown below. This information was collected using the PhyTip ME 1000 and ME 200 Purification Systems.

IMAC Wash Buffer I is labeled and supplied as a concentrated solution of Phosphate Buffer containing Imidazole. Recommended procedure is to dilute the buffer 20 X for use in the purification process, and higher concentrations should be evaluated for their effect on final purity. By varying the dilution of this buffer from 20 X to 1 X, purity and yield of final product may change. We recommend you begin with the lowest concentration of buffer and increase as needed to obtain the desired purity. As you increase the concentration of the Wash buffer there may be a reduction in yield of target protein.

IMAC Elution Buffer, as supplied, contains: 10 mM NaH2PO4, 0.3M NaCl and 200 mM Imidazole, pH7.4.

Documents
Robotics Systems Compatibility

His-tagged Proteins PhyTip Columns are available for these robotic systems.



Part Numbers

Ni-IMAC PhyTip Columns