This video will show the isolation of IGY antibodies from hens, which can be immunized either with the gene gum, plasmid, or more traditionally using intramuscular injection action. First, the egg yolk is isolated. Then the lipids are precipitated, followed by a precipitation of a protein fraction containing the antibodies which are isolated in a further precipitation step.
Finally, the antibodies are dialyzed against PBS.Yes.Hello. My name is Ard. That is my colleague, the Adema, and we work here in the very old and famous Institute of Pharmacology, which belongs to the SHA University Medicine of Berlin.
And in the following, we would like to show you the extraction of IGY from chicken, egg, yogurt, and you think a very and very simple and also very cost effective method. And altogether we belongs to the so-called IGY technology. The first step of IGY preparation is the isolation of the egg yolk.
For this procedure, we need tube racks, blue caps filter paper, pipette tips buffer, a glass beaker, a spoon for the separation of the yolk from the egg white, and of course, the eggs. The egg is cracked open above the yolk spoon to discharge the egg white. Then the yolk spoon containing the egg is gently agitated to remove most of the egg white.
The yolk is then moved on to filter paper to remove egg white proteins as completely as possible. The egg skin is very stable in eggs, no older than one month after storage longer than three months. The yolk skin may rupture due to the aging process during rolling on filter paper.
Thereafter, the yolk skin is opened with a pipette tip and transferred to a tube. Finally, you see that the yolk has a volume of approximately 15 milliliters. That volume is normal for an adult laying hen and varies only minimally.
The yolk volume is recorded 3.5%Polyethylene glycol is used as a precipitant in PBS buffer to separate the yolk proteins, including IGY, from the lipids in the egg yolk, 1.58 grams of polyethylene glycol, which corresponds to 3.5%of the final yolk and buffer volume is weighed out on a balance scale. The 15 milliliter yolk is mixed with twice the volume of PBS buffer, so 30 milliliters of PBS is added to a final volume of 45 milliliters. The polyethylene glycol is then added to the mixture.
The tube is capped and then vortex thoroughly for 20 to 30 seconds. Periodically invert the tube. During the vortexing procedure.
After vortexing, the yolk should be fully in suspension. The tubes are now placed on a roller mixer for 10 minutes. The result is a homogenous mixture with a changed color from yellow to light yellow.
Now the mixture is centrifuged for 20 minutes. The centrifugation separates a clear sup, natant containing IGY, and other proteins from a yellow precipitate, which consists mainly of lipids. In the next step, the S supernatant is poured through a folded filter and transfer to a new tube.
The new volume is recorded and the precipitate is discarded. Now, once again, polyethylene glycol is added to the supernatant in a final concentration of 12%That means 3.5%in the first precipitation, plus 8.5%of the resulting SNAT volume making 12%Thereafter, the solution is stirred incubated on a roller mixer as before and centrifuged for 20 minutes. Now, the polyethylene glycol precipitates the IGY and other proteins.
After this centrifugation step, the pellet will be used in preparation for dissolving. The pellet glass sticks are disinfected with 70%ethanol while the sticks dry, the S supernatant is discarded. The tube is then inverted to drain off the last of the supinate.
One milliliter of PBS is then added to the pellet. The pellet is dissolved using the glass stick while thoroughly vortexing. Now you see a slightly yellow solution.
Now, PBS is added to a final volume of 10 milliliters followed by a third polyethylene glycol precipitation in a concentration of 12%as before in contrast to the second centrifugation. This time a white precipitate is obtained after centrifugation for 20 minutes. The pellet is dissolved in the same way as before, but in two steps.
In the first step, it is dissolved in 0.8 milliliters of PBS and transferred in a microdialysis capsule. Then the tube is rinsed with an additional 0.4 milliliters of PBS, which is also transferred to the capsule. In the next step, a dialysis bag is cut with disinfected scissors and rinsed with distilled water.
The dialysis bag is then placed on the lower part of the capsule and shut tightly by the upper part. Finally, the capsules are transferred to a glass beaker placed on a magnetic stir. The IGY solution is dialyzed overnight against 0.1%saline.
The next day, the IGY solution is dialyzed for three hours against PBS. After finishing the dialysis, the dialysis membrane is punched with a pipette tip and the IGY solution is removed and transferred to a small tube. The final volume is recorded.
The final product is a clear solution, which can be stored at minus 20 degrees Celsius for several years. According to our experience, do not store the IGY preparation at minus 70 degrees Celsius since we observed a significant loss of activity at this temperature. Well, I hope we were able to convince you that IDY extraction from a is a really simple and effective procedure when following our protocol.
The equipment needed as minimal, and we believe there is a sound ratio between effort and benefit. I hope that this video publication will encourage you to make good use of our protocol and also technology, as I will. Thank you.
