Set Up:

1. Order all needed materials

2. Set up 4 vials with regular fruit fly food to serve as the control

3. Set up 2 vials with 3 milligrams of caffeine mixed in with food (use gloves and face mask when handling caffeine)

4. Set up 2 vials with 0.3 milligrams of nicotine mixed in with food (use gloves and face mask when handling nicotine)

5. Release 10 genetically modified fruit flies with Parkinson’s Disease into 2 vials of regular food, 1 vial with caffeine, and 1 vial with nicotine

6. Repeat step 5 with regular fruit flies

7. Allow fruit flies to live in these habitats for a minimum of 3 weeks

8. Take out 10 flies from each vial and add them into separate test tubes with distilled water

9. Smash the flies with a pestle

10. Place them into microtubes. Label the microtubes with what type of fruit fly it is (with PD or without) and what food it was fed (regular or with caffeine)

11. Preparing ELISA Kit plate for Alpha Synuclein Test:

12. Add 50 mL of the standards/controls into the wells of the plate

13. Create experimental wells and add 50 mL of fruit flies to the wells

14. Add 50 mL of the A-Synuclein Detection solution into the standard and experimental wells, tap the side of the plate to mix

15. Cover the plate and incubate for 3 hours at room temperature

16. Aspirate the solution and wash wells with wash buffer 4 times

17. Add 100 mL of Anti Rabbit IgG into all except chromogen banks, including the empty rows in which chromogen will be added

18. Cover the plate and incubate for 30 minutes at room temperature

19. Thoroughly aspirate the solution and wash wells four time with wash buffer

20. Add 100 ml of stabilized chromogen to each well, including the chromogen banks. Substrate solution will turn blue.

21. Incubate for 30 minutes at room temperature in the dark

22. Add 100 ml of stop solution to each well, tap side of plate to mix, solution will  turn from blue to yellow

23. Place into plate reader and collect data about absorption rates

24. Preparing ELISA Kit plate for Tau Test:

25. Add 100 mL of the standards/controls into the wells

26. Create experimental wells and add 100 mL of fruit flies to the wells

27. Cover and incubate for 2 hours at room temperature

28. Thoroughly aspirate the solution and wash wells four times with wash buffer

29. Add 100 ml of Tau detection antibody into each well except chromogen banks

30. Cover plate and incubate for 1 hour at room temp

31. Thoroughly aspirate the solution and wash wells four time with wash buffer

32. Add 100 mL of Anti Rabbit IgG into all wells except chromogen banks

33. Cover the plate and incubate for 30 minutes at room temperature

34. Thoroughly aspirate the solution and wash wells four times with wash buffer

35. Add 100 ml of stabilize chromogen to each well, including chromogen banks. Substrate solution will turn blue.

36. Incubate for 30  mins at room temp in the dark

37. Add 100 ml of stop solution to each well, tap side of plate to mix, solution will turn from blue to yellow

38. Place into plate reader and collect data about absorption rates

39. Analyze data