PhyNexus IMAC (Immobilized Metal Affinity Chromatography) PhyTip® columns purify or immobilize functional, recombinant His tagged protein or antibody quickly at high purity and concentration. IMAC separation resin in pipette tips is used to generate highly enriched sample from microliter to milliliter volumes in a reproducible, automatable format. The unique PhyTip column design allows bidirectional flow of the sample through the column to increase capture and elution of the target protein, maximizing the use of the resin and leading to the highest yield possible.

  • IMAC (Ni-IMAC) affinity columns routinely produce samples of greater than 90% purity. The purity is demonstrated by the absence of background proteins.
  • Recover 60-80% of fully functional protein with greater than 90% purity
  • Process small sample volumes in a reproducible, high throughput automation
  • IMAC(Ni-IMAC) tips are processed in a high throughput automation
  • Elution volumes as low as 10 μL, producing enrichment factors as high as 50 fold, with concentrations of purified protein of up to 10 mg/mL

IMAC columns with our proprietary Dual Flow Chromatography provide complete antibody binding and high final concentrations. PhyTip columns are compatible with most 8, 12, and 96-channel automation liquid handling robots including Agilent TechnologiesBeckman CoulterDynamic DevicesPerkin ElmerHamilton, and Tecan.

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
Compatibility

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

Citations

Publications


Discovery of specific antigens that can predict microfilarial intensity in Loa loa infection.
Drame P, Bennuru S, Nutman T
Publication: Journal of clinical microbiology, 2017 pp: JCM.00513-17
doi: 10.1128/JCM.00513-17
www.ncbi.nlm.nih.gov/pubmed/28637911

Plant cell wall glycosyltransferases: High-throughput recombinant expression screening and general requirements for these challenging enzymes
Welner D, Shin D, Tomaleri G, DeGiovanni A, Tsai A, Tran H, Hansen S, Green D, Scheller H, Adams P
Publication: PLOS ONE Editors: Hofmann A, 2017 vol: 12 (6) pp: e0177591
doi: 10.1371/journal.pone.0177591
dx.plos.org/10.1371/journal.pone.0177591

The Type IV Pilus Assembly ATPase PilB of Myxococcus xanthus interacts with the Inner Membrane Platform Protein PilC and the Nucleotide Binding Protein PilM
Bischof L, Friedrich C, Harms A, Søgaard-Andersen L, van der Does C
Publication: Journal of Biological Chemistry, 2016 (6) pp: 25
doi: 10.1074/jbc.M115.701284
www.jbc.org/lookup/doi/10.1074/jbc.M115.701284

Production of Site-Specific Antibody–Drug Conjugates Using Optimized Non-Natural Amino Acids in a Cell-Free Expression System
Zimmerman E, Heibeck T, Gill A, Li X, Murray C, Madlansacay M, Tran C, Uter N, Yin G, Rivers P, Yam A, Wang W, Steiner A, Bajad S, Penta K, Yang W, Hallam T, Thanos C, Sato A
Publication: Bioconjugate Chemistry, 2014 vol: 25 (2) pp: 351-361
doi: 10.1021/bc400490z
dx.doi.org/10.1021/bc400490z

Fluorescent isotope-coded affinity tag 2: Peptide labeling and affinity capture
Rivera-Monroy Z, Bonn G, Guttman A
Publication: ELECTROPHORESIS, 2009 vol: 30 (7) pp: 1111-1118
doi: 10.1002/elps.200800830
dx.doi.org/10.1002/elps.200800830

A simple high-throughput purification method for hit identification in protein screening
Cummins E, Luxenberg D, McAleese F, Widom A, Fennell B, Darmanin-Sheehan A, Whitters M, Bloom L, Gill D, Cunningham O
Publication: Journal of Immunological Methods, 2008 vol: 339 (1) pp: 38-46
doi: http://dx.doi.org/10.1016/j.jim.2008.07.016
www.sciencedirect.com/science/article/pii/S002217590800238X

Optimisation of a simple method to transiently transfect a CHO cell line in high-throughput and at large scale
Mark Abbott W, Middleton B, Kartberg F, Claesson J, Roth R, Fisher D, AstraZeneca
Publication: Protein Expression and Purification, 2015
doi: 10.1016/j.pep.2015.08.016
https://www.ncbi.nlm.nih.gov/pubmed/26291269

Widening the bottleneck: Increasing success in protein expression and purification
Hopkins R, Esposito D, Gillette W, SAIC-Frederick I
Publication: Journal of Structural Biology, 2010 vol: 172 (1) pp: 14-20
doi: 10.1016/j.jsb.2010.07.005
https://www.ncbi.nlm.nih.gov/pubmed/20650317

Discovery of a potent class of PI3K α inhibitors with unique binding mode via Encoded Library Technology (ELT)
Yang H, Medeiros P, Raha K, Elkins P, Lind K, Lehr R, Adams N, Burgess J, Schmidt S, Knight S, Auger K, Schaber M, Franklin G, Ding Y, Delorey J, Centrella P, Skinner S, Clark M, Cuozzo J, GlaxoSmithKline
Publication: ACS Medicinal Chemistry Letters, 2015 pp: 1-17
doi: 10.1021/acsmedchemlett.5b00025
https://www.ncbi.nlm.nih.gov/pubmed/26005528

Select your resin, robotic system, tip and resin volume and see the results in the table below.

IMAC PhyTip Columns