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 Devices,  Hamilton, Opentrons, Perkin Elmer, and Tecan.

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.

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

Protein G Columns