Protein G PhyTip Columns

Protein G PhyTip Columns demonstrate high binding affinity and high selectivity affinity purification of functional antibodies (IgGs).  They 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

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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
Robotic Systems Compatibility

Protein G PhyTip Columns are available for these robotic systems.

Part Numbers

Protein G PhyTip Columns