Protein G PhyTip Columns demonstrate high binding affinity and high selectivity affinity purification of functional antibodies (IgGs). Protein G columns are shown to have high selectivity for varying isotypes and species of IgG and obtain highly concentrated purified proteins

  • High binding affinity
  • High selectivity for different species and isotypes of antibody
  • 70-90% IgG recovery

Protein G tips 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 with Protein G

PhyTip columns with Protein G 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

All PhyTip columns with Protein G are supplied with recommended PhyTip buffers including: Capture Buffer – provided for those situations where additional buffer needs to be added to supplement the volume of the sample and to ensure correct pH for capture Wash Buffer I – Phosphate Buffer solution pH 7.4 Wash Buffer II – Saline solution. NOTE: no buffering capacity so as to ensure effective elution Enrichment Buffer – for the final elution step – Phosphate Buffer solution pH 2.5 Neutralization Buffer. – Tris Buffer solution pH 9.0

Note: Enrichment buffer is supplied as 4 mL of a pH 2.5 Phosphate buffer solution, if protein to be purified requires less acidic elution conditions e.g. pH 2.8, the enrichment buffer pH can be changed as follows: Take 1 mL of standard Enrichment Buffer (pH 2.5) and add 30 μL of 1 M Tris Buffer standard Neutralization buffer to obtain 1 mL of pH 2.8 elution buffer (actual pH may vary depending upon volumetric accuracy) For a pH 3.0 enrichment buffer, take 1 mL of standard Enrichment Buffer (pH 2.5) and add 40 μL of 1 M Tris Buffer standard Neutralization Buffer to obtain 1 mL of pH 3.0 elution buffer (actual pH may vary depending upon volumetric accuracy)

For the neutralization step add 25% v/v of the elution volume e.g. if the elution volume is 20 μL add 5 μL of 1 M Tris Neutralization Buffer

200+ PhyTip columns with Protein G resin:

For a 200 μL sample with 5 μg IgG2a (anti-FITC MAb.) containing 1 mg BSA, processed using the conditions shown below, greater than 40% of the original IgG mass is recovered in the final sample volume. In addition, the recovered IgG is over 95% pure as determined by SDS-PAGE with Coomassie detection.

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

Purify: 200 μL of PhyNexus Protein G 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 Wash Buffer II, passed over the resin bed for two cycles at a flow rate of 500 μL/min.

Enrich: elute the protein into solution with 10 μL of PhyNexus Protein G Enrichment Buffer, passed over the resin bed for five cycles at a flow rate of 500 μL/min. Neutralize with 3 μL of PhyNexus Protein G Neutralization Buffer.

Protein G Enrichment Buffer as shipped contains: 111mM NaH2PO4, 140mM NaCl in 14.8mM H3PO4, pH 3.0

1000+ PhyTip columns with Protein G resin:

For a 500 μL sample with 10μg IgG2a (anti-FITC MAb.) containing 5 mg BSA, processed using the conditions shown below, greater than 40% of the original IgG mass is recovered in the final sample volume. In addition, the recovered IgG is over 95% pure as determined by SDS-PAGE with Coomassie detection.

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

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

Enrich: elute the protein into solution with 15mL of PhyNexus Protein G Enrichment Buffer, passed over the resin bed for five cycles at a flow rate of 500 μL/min. Neutralize with 5 μL of PhyNexus Protein G Neutralization Buffer.

Application

PhyTip columns are unique capture, purification and enrichmentTM 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.

Data

PhyNexus recommends the use of the PhyTip Starter Kit containing IgG as a standard to verify the use of PhyTip columns with all applications.

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.

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.

PhyTip columns with Protein G are stored in Glycerol when shipped from PhyNexus.

Documents
Compatibility

Protein G PhyTip Columns are available for these robotic systems.

Citations

Publications


Facile fabrication and instant application of miniaturized antibody-decorated affinity columns for higher-order structure and functional characterization of TRIM21 epitope peptides
Al-Majdoub M, Opuni K, Koy C, Glocker M, University Medicine Rostock
Publication: Analytical Chemistry, 2013 vol: 85 (21) pp: 10479-10487
doi: 10.1021/ac402559m
https://www.ncbi.nlm.nih.gov/pubmed/24094071

Agonists and Antagonists of Protease-Activated Receptor 2 Discovered within a DNA-Encoded Chemical Library Using Mutational Stabilization of the Target
Brown D, Brown G, Centrella P, Certel K, Cooke R, Cuozzo J, Dekker N, Dumelin C, Ferguson A, Fiez-Vandal C, Geschwindner S, Guié M, Habeshian S, Keefe A, Schlenker O, Sigel E, Snijder A, Soutter H, Sundström L, Troast D, Wiggins G, Zhang J, Zhang Y, Clark M
Publication: SLAS DISCOVERY: Advancing Life Sciences R&D, 2018 pp: 247255521774984
doi: 10.1177/2472555217749847
journals.sagepub.com/doi/10.1177/2472555217749847

Auto-induction of Pichia pastoris AOX1 promoter for membrane protein expression
Lee J, Chen H, Liu A, Alba B, Lim A
Publication: Protein Expression and Purification,  2017
doi: 10.1016/j.pep.2017.06.006
www.sciencedirect.com/science/article/pii/S1046592817300669

Solid-State Hydrogen–Deuterium Exchange Mass Spectrometry: Correlation of Deuterium Uptake and Long-Term Stability of Lyophilized Monoclonal Antibody Formulations
Moorthy B, Zarraga I, Kumar L, Walters B, Goldbach P, Topp E, Allmendinger A
Publication: Molecular Pharmaceutics, 2017 pp: acs.molpharmaceut.7b00504
doi: 10.1021/acs.molpharmaceut.7b00504
pubs.acs.org/doi/abs/10.1021/acs.molpharmaceut.7b00504

Phage display and hybridoma generation of antibodies to human CXCR2 yields antibodies with distinct mechanisms and epitopes
Rossant C, Carroll D, Huang L, Elvin J, Neal F, Walker E, Benschop J, Kim E, Barry S, Vaughan T
Publication: mAbs, 2014 vol: 6 (6) pp: 1425-1438
doi: 10.4161/mabs.34376
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4622561/

Posters


Screening for Chromatographic Success and Failure on Small Scale
W. Gillette (NCI/SAIC-Frederick)

Space is the Place: Using DOE and Microscale Techniques to Define Process Boundaries
M. Wenger (Merck & Co.)

Development of Small-Scale, High Throughput, Chromatographic Protein Purification
K. Valente1, M. Wenger2, J. Shanter1, S. Sagar1, J. Konz1 (1Merck Biopurification Development, 2Merck Bioprocess and Bioanalytical Research)

Accelerated antigen identification: a case study
Raven biotechnologies, inc.

An Improved Method for Chromatin Immunoprecipitation Using Micro-Scale Affinity Columns
H. Li, J. Sklar (Yale School of Medicine); C. Hanna, J. Lambert (PhyNexus)

Optimization of protein purification using micro-scale separation columns
J. Lambert, M. Anderson, D. Gjerde, L. Hoang (PhyNexus)

A flexible platform for automated high-performance protein purification using micro scale separation technology
J. Lambert, M. Anderson, D. Gjerde, L. Nguyen (PhyNexus)

A novel approach to high-throughput monoclonal and recombinant antibody enrichment and characterization
J. Lambert, C. Hanna, U. Banik (PhyNexus); D. Sexton, K. Kopacz (Dyax); S. Wiltshire (HTS Biosystems)

High-throughput sample processing and affinity characterization of antibodies and recombinant proteins
S. Wiltshire, J. Lambert (HTS Biosystems); C. Hanna (PhyNexus)

Innovative Solutions for Automated Protein and Nucleic Acid Purification
Lee Hoang, PhyNexus, Inc., PhyNexus Webinar

Optimizing Efficiency and Effectiveness When Expressing Proteins for Drug Discovery
Krista Bowman, Genentech, Inc., Protein Engineering Summit (PEGS) 2012

Automated, High Throughput Protein Purification and Sample Prep Using PhyTip Columns for Therapeutic Leads Screening and Process Development
Lee Hoang, PhD., PhyNexus, Inc., The 5th Annual Proteins Congress 2012, London

Identification of Unknown Target Antigens Using PhyNexus Technology
Claudia Fieger, Tim Hotaling, MarcroGenics, Inc.

Optimization of Protein Purification Using Small-Scale Separation Columns
Chris Suh, PhD., PhyNexus, Inc., Protein Therapeutics Discovery and Development

PhyNexus Users Group Symposium 2014
Douglas Gjerde, PhyNexus
Lee Hoang, PhyNexus
Alice Yam, Sutro

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

Protein G Columns