Protein A PhyTip Columns demonstrate high binding affinity and high capacity affinity purification of functional antibodies (IgGs).

PhyNexus has developed a unique technology for the micro scale purification of engineered proteins and antibodies which allow researchers to routinely purify and enrich sample volumes up to 1 mL. The exclusive design of the PhyTip™ columns allow for elution volumes as low as 10μL, thus producing enrichment factors as high as 50 x, with concentrations of purified protein of up to 5 mg/ml.

The process to purify and enrich is a simple three step technique where the protein of interest is first captured, then purified and finally enriched. The entire process can take less than 15 min to produce high concentrations of fully functional protein ready for further analysis.

  • 70-90% IgG recovery
  • Obtain highly concentrated purified proteins
  • Protein A PhyTip columns are shown to extract IgG proteins with very high purity from varying levels of background proteins

Protein A 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 Technologies, Beckman Coulter, Dynamic Devices,  Hamilton, Opentrons, Perkin Elmer, and Tecan.

Related: Protein G ProPlus/ProPlus LX  CaptureSelect AffinityCustom Resins

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Protein-A-PhyTip-Columns

Protein A tips are available in tip volumes (µL) of 200|300|500|1000 and resin volumes (µL) of 5|10|20|40|80|160|320 depending upon robot configuration.

Protein A PurificationLane 1: marker, 2: 2 µg BSA, 3: 2 µg IgG2a, 4, 5: Protein A resin only, respectively; 6, 7, 8: 2 µl each of Protein A purified IgG2a from PBS, PBS containing 5 mg BSA (2 and 5 cycles capture), respectively; 9, 10, 11: 2 µl each of Protein A purified IgG2a from PBS, PBS containing 5 mg BSA (2 and 5 cycles capture), respectively.
Protein A Purification PhyNexus Dual Flow Chromatography Affinity purification of antibody sample Protein A Binding

PhyTip columns with Protein A have been optimized for use with specific PhyNexus reagents and instrument flow rates/volumes as shown below. This information was collected using the PhyNexus Purification Systems. All PhyTip columns with Protein A 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 A 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 A 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 A 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 A 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 A 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 A 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 A Neutralization Buffer.

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

Protein A Columns