Membranes ELISA Protocol for use with GPCR Polyclonal Antibodies from Proteimax

Buffers to prepare for the ELISA:

Wash Buffer (10X PBS):
To prepare 1 literof 10X PBS, first dissolve80 g NaCl, 2 g KCl, 14.4 g Na₂HPO₄, and 2.4 g KH₂PO₄.in 800 mL distilled water. Adjust pH to 7.4. Then bring to 1000 mLwith distilled water. Autoclave.

Blocking Buffer:
1X PBS with 1% BSA, 5% sucrose, and 0.05% sodium azide. Store at 4°C.
Note: Do not add sucrose to the blocking buffer for use with membranes.

Treatment Buffer:
50mM Tris-Base, pH 7.4

Fixing Buffer:
3.7% Formaldehyde in 1X PBS

Developing Buffer:
• 100 mM Tris-Base
• 4 mg/mL Sigma 104 Phosphatase Substrate (p-Nitrophenyl phosphate, disodium).
For 10 mL of Developing Buffer, mix 5 mL of 100 mM Tris with 5 mL of Sigma 104. Do not pH adjust this solution.

ELISA Protocol:

1. Dissolve the membranes in 1X PBS (0.05 µg/µL) so that  100 µL of solution will have 5 µg of membranes.
2. Coat the 96-well plates (High Binding non-sterile 96-well EIA/RIA plate flat bottom without lid (Corning/Costar, Cat. No. 3590) with 5 µg of membranes (100 µL/well).
3. Allow the membranes to dry out at room temperature (it takes about 48 hours).
4. It is possible to store the dried plates for several months.
5. Warm 1X PBS and Treatment Buffer to room temperature (RT).
6. Dissolve the drug of interest in Treatment Buffer.
7. Wash the membranes once with 1X PBS (150 µL/well).
8. Add either Treatment Buffer alone for control membrane proteins (just secondary antibody wells and primary/secondary antibodies wells without drug treatment), or Treatment Buffer with drugs of interest.  All experiments should be performed in triplicates.
9. Incubate at 37°C for 30 minutes to 1 hour.
10. Remove activation or stimulation media manually or by aspiration.
11. Immediately (row by row) fix membranes with 3.7% Fixing Buffer. Note: Prepare fresh Fixing Buffer. Using a multi-channel pipettor, add 150 µL/well of Fixing Buffer (RT) and allow incubation on bench top for 20 minutes at RT with no shaking. Make sure to carefully add the solution down the sides of the wells to avoid detaching the membranes from the well bottom.
12. Remove Fixing Buffer by aspiration to an appropriate waste container.
13. Using a multi-channel pipettor, add 200 µL of 1X PBS. Note: Make sure to carefully add the solution down the sides of the wells to avoid detaching the membranes from the well bottom.
14. Wash five times with 1X PBS for 5 minutes per wash.  Washing should be done with shaking on a rotator for 5 minutes at RT.  Always remove wash solution by aspiration.  Note: Do not allow membranes/wells to become dry during washing.  Immediately add the next wash after aspiration.
15. Using a multi-channel pipettor, block membranes/wells by adding 150 µL/well of Blocking Buffer. Add the solution carefully by pipetting down the sides of the wells to avoid detaching the membranes from the well bottom.
16. Allow blocking for 90 minutes at RT with moderate shaking on a rotator.
17. Remove the blocking buffer by aspiration.
18. Immediately add the primary antibody diluted in 1X PBS (100 µL/well).  Do not forget to leave 3 wells just for secondary antibody. Each primary antibody requires a specific dilution, refers to the antibody manual for the recommended starting dilution.
19. Incubate with primary antibody overnight at 4°C.
20. Wash 3 times with 1X PBS for 5 minutes per wash. Washing should be done with shaking on a rotator for 5 minutes at RT. Using a multi-channel pipettor, add 200 µL/well of 1X PBS. NOTE: Make sure to carefully add the solution down the sides of the wells to avoid detaching the membranes from the well bottom.
21. Dilute the Alkaline Phosphatase labeled secondary antibody in 1X PBS (use the recommended dilution from the manufacturer).
22. Add secondary antibody diluted in 1X PBS (100 µL/well).
23. Incubate for 120 minutes with gentle shaking at RT.  Protect plate from light during incubation.
24. Wash 4-5 times (depends on the secondary antibody manufacturer) with 1X PBS for 5 minutes per wash.  Washing should be done with shaking on a rotator for 5 minutes at RT.  Using a multi-channel pipettor, add 200 µL/well of 1X PBS.  Note: Make sure to carefully add the solution down the sides of the wells to avoid detaching the membranes from the well bottom.
25. After final wash, remove wash solution completely from wells. Turn the plate upside down and tap or blot gently on paper towels to remove traces of wash buffer. Add immediately the Developing Buffer (100 µL/well).
26. Read the plate at 400-420 nm in 5 minute intervals for 40 minutes.  Use as your result the inclination of the linear part of your curve. Subtract the secondary antibody recognition and use the secondary and primary result (without treatment) as 100%.