ELISA Procedure

Materials

• Protease inhibitor cocktail
• Standard Diluent Buffer
• α-Synuclein standard
• Working Washing Buffer
• Detection Antibody
• Anti-rabbit 1gG Horseradish Peoxidase (HRP)
• wash buffer
• stabilized chromogen
• stop solution
• α-Synuclein antibody-coated wells
• plate covers
• 96-well frame
• pipette
• ELISA plate reader

Procedure

Handling of Assay Kit

• Stored at 4°C when not used
• All components were warmed to room temperature before being used in experiment
• Cell Extraction Buffer thawed on ice prior to use
• All reagents were covered or capped when not in use

1. The number of α-Synuclein antibody-coated wells was determined and were inserted into the 96-well frame.

2. 100 µl of Standard Diluent Buffer was added to wells. Two wells were left empty for chromogen blanks.

3. Standards were prepared as described below:

Vial	Standard	Alpha-Synuclein Standard (µl)	Diluent Buffer (µl)
A	100 ng/ml	As described on vial	As described on vial
B	50 ng/ml	250 µl of A	250 µl
C	25 ng/ml	250 µl of B	250 µl
D	12.5 ng/ml	250 µl of C	250 µl
E	6.25 ng/ml	250 µl of D	250 µl
F	3.13 ng/ml	250 µl of E	250 µl
G	1.56 ng/ml	250 µl of F	250 µl
H	0 ng/ml	0 µl	250 µl

4. 100 µl of standard, samples, or controls was added to the wells in duplicate. The side of the frame was tapped for gentle mixture of solutions. Samples were diluted at a 1:10 ratio with Standard Diluent Buffer (10 µl of sample and 90 µl of Buffer).

5. Wells were covered with plate covers and were left stationary at room temperature for two hours.

6. After two hours, the solution was poured out from the wells and the wells were washed four times.

Washing Summary

• Solution was removed from wells over sink.
• Wells were filled to the top with Working Washing Buffer.
• Wells soaked for 15-30 seconds.
• Liquid was poured out and the above steps were repeated three more times.
• Plate was dried by tapping out on paper towel.

7. 100 µl of Detection Antibody for α-Synuclein was pipetted into each well except the chromogen blanks. Frame was tapped gently to mix wells.

8. Wells were covered with plate covers and were left stationary at room temperature for one hour.

9. Plates were washed as described above.

10. 100 µl of Anti-rabbit 1gG Horseradish Peoxidase was added to each well except for the chromogen blanks.

11. Wells were covered with plate covers and were left stationary at room temperature for thirty minutes.

12. Plates were washed as described above.

13. 100 µl of stabilized chromogen was added to each well and a change in colour from clear to blue was observed.

14. Wells were covered with plate covers and were left stationary at room temperature for 30 minutes in the dark.

15. 100 µl of stop solution was added to each well. Frame was tapped gently to mix wells. A change in colour from blue to yellow was observed.

16. Absorbance of each well was read at 450 nm. The chromogen blanks were used as the blank on the ELISA microplate reader.

17. The amount of α-synuclein protein in each treatment was determined by plotting the standard curve with Graph Pad Prism and performing nonlinear regression. The concentration of the unknown samples could then be determined from the standard curve graph.

Pictures from Procedure (click on each picture to open it in its own window)

ELISA Alpha-Synculein Kit

Set up of area prior to ELISA

Patrick performing the ELISA

Washing the well plates in between steps