Aug 26, 2020
Purification of avian egg yolk immunoglobulins using the water dilution method, precipitation with isopropanol and using HiTrap™ Columns.
- 1University of the West Indies St. Augustine
- University of the West Indies
- angel.vaillant@sta.uwi.edu
Protocol Citation: Angel A Justiz-Vaillant 2020. Purification of avian egg yolk immunoglobulins using the water dilution method, precipitation with isopropanol and using HiTrap™ Columns.. protocols.io https://dx.doi.org/10.17504/protocols.io.bkbnksme
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 26, 2020
Last Modified: August 26, 2020
Protocol Integer ID: 41038
Separate egg yolk from the egg white.
Add nine parts of distilled water to one part egg yolk.
Mix and stir slowly for 6.30 h at 4°C.
Centrifuge at 10 000 × g, at 4°C for 25 min to precipitate the lipids.
Collect the supernatant containing the IgY (water soluble fraction).
Slowly add 5 ml of cold isopropanol to the preparation, stirring constantly, and centrifuge at low spead.
Dilute the precipitate in PBS and adjust pH to 7.6. Calculate the protein concentration by ELISA. spectrophotometry or Bradford method.
Determine the purity of the preparation by SDS-PAGE.
Fill the syringe or pump tubing with de-ionized water. Remove the stopper and connect HiTrap™ column to syringe (use the connector supplied).
Snap off tab on the column outlet.
Wash out the ethanol with 26 ml of de-ionized water.
Equilibrate column with 26 ml of binding buffer. The recommended flow rate is 5ml/min.
Apply the IgY sample using a syringe fitted to Luer connector or by pumping it onto the column.
For better results, use a flow rate of 0.5 to 5.1 ml/min during sample application.
Wash with at least 51 ml of binding buffer or no material remains in the effluent.
Maintain a flow rate of 5 to 11 ml/min for washing.
Elute with 51 ml of elution buffer using a one-step or using a linear gradient though larger volumes are often required to break the interaction.
After elution, regenerate the column by washing with 36 ml of wash buffer.
Re-equilibrate the column with 26 ml of binding buffer.
The column is now prepared for a new purification.