Immunology Field Trip

I. Purification of IgY Antibodies from Egg Yolks

Equipment and Reagents:

Protocol

1. Students should label two 1.5ml Eppendorf tubes with their initials.
2. Place a folded paper towel into a weigh boat. This is for the egg yolk.
3. Crack a room temperature egg over the 1 L glass beaker. Saving the yolk, and separate the egg white into the beaker. Place the yolk on the paper towel.
4. Using a transfer pipet, puncture the yolk and take up 2ml of yolk and add it to the tube containing 8 mls of Precipitation Buffer A. (10 ml total volume)
5. Cap the tube and gently mix the yolk with the buffer by inverting the tube for 2 minutes.
6. Place the gauze pad over the top of the 100ml beaker to form a filter. Filter the mixture by carefully pouring the contents of the tube through the gauze and into the beaker.
7. After the filtering, remove the gauze to the 1 L glass beaker.
8. Using a P1000 Pipetman and blue tip, add 1 ml of the filtered yolk mixture into one (1) labeled 1.5ml Eppendorf tube.
9. Microfuge the tube in a balanced, tabletop microfuge for 10 minutes.
10. Carefully remove the tubes from the centrifuge so that the pellet is not disturbed. Pipet 500µl of the supernatant from the top of the tube into the clean, labeled Eppendorf tube.
11. With a P1000 and a clean pipet tip, add 100µl of Precipitation Buffer B to the 500µl of supernatant.
12. Cap the tube and gently mix by inverting the tube for 2 minutes.
13. Centrifuge for 5 or 10 minutes.
14. Without touching the pellet, use a P1000 to pipette off most of the supernatant. Discard the tip and the liquid in the waste beaker.
15. Use a P200 and a yellow tip to carefully remove any remaining liquid from the tube.
16. Add 1 ml of 1X PBS (from the 50 ml tube) to resuspend the pellet by performing the following steps in order:
    1. Add 500μl PBS to the antibody-containing pellet using a p1000 and a blue tip
    2. Use the tip of the pipette to scrape at the pellet (or the place where the pellet should be) to release it from the side of the tube. Use the pipette to tip mix the solution by drawing and dispensing several times.
    3. Add another 500μl PBS to the tube and cap it.
    4. Vortex the tube for ten to fifteen seconds.

II. Using Purified IgY to Detect Proteins

Equipment and Reagents:

Protocol

Note: Always wear gloves and use forceps when handling nitrocellulose membranes. This membrane has been dot blotted with proteins from E. coli, Salmonella and a negative control.

1. Label the weigh boat containing the membrane with your initials.
2. Add 5 ml PBS + 3% BSA (Blocking Solution) to the membrane and incubate at room temperature with gentle agitation for at least 10 mins.
3. Pour the Blocking Solution back into its tube; leave the membrane in the weigh boat.
4. Pipet 1ml IgY antibodies onto membrane and incubate on a rocker for 15 mins.
5. Pour off the IgY solution into the waste beaker.
6. Wash the membrane by pouring about 10 ml of PBS onto it; gently shake for 2 mins.
7. Pour the PBS into the waste beaker and repeat the wash with another 10 ml PBS.
8. Pour the PBS into the waste beaker and add 4ml HRP-conjugated, goat anti-IgY.
9. Incubate the membrane and this secondary antibody on a rocker for 15 mins.
10. Pour the secondary antibody back into its tube.
11. Wash the membrane by pouring about 10 ml of PBS onto it; gently shake for 2 mins.
12. Pour the PBS into the waste beaker and repeat the wash 2 more times with 10 ml PBS.
13. Pour the PBS into the waste beaker and pipet 750µl of the TMB Substrate onto the membrane.
14. Allow the color to develop, then pour off the substrate solution.
15. Quickly rinse the membrane twice with ddH₂O to completely remove the substrate.
16. Dry the membrane; wrap the blot in plastic wrap to preserve it. The color bleaches if the filter is not storred in the dark.

Note: Blocking solution and the AP-conjugated antibody can be used up to five times.  Store Blocking solution in the freezer and the antibody in the refrigerator.