Cell ELISA Protocol for use with GPCR Polyclonal Antibodies from Proteimax

Date Published: July 18, 2023

Note:The following protocol has been developed for use with GPCR Polyclonal Antibodies manufactured by Proteimax. Other antibodies have not been tested using this protocol. For additional assistance, please contact Technical Support.

Buffers to prepare:

Wash Buffer (10X PBS): To prepare 1 liter of 10X PBS, first dissolve 80 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 mL with 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:
50 mM 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 10mL of Developing buffer, mix 5 mL of 100 mM Tris with 5 mL of Sigma
104. Do not pH adjust this solution.

Protocol:

  1. Allow SKNSH cells (ATCC; HTB-11) to grow in a T75 flask using standard tissue culture procedures until cells reach near confluency (~1.2 x 107 cells; D-MEM, 10% FBS; Gibco).
  2. Remove growth media, wash cells with sterile 1X PBS, and trypsinize cells for displacement.
  3. Neutralize displaced cells with culture media and clarify by centrifugation (500 x g).
  4. Remove supernatant and disrupt the cell pellet manually by hand tapping the collection tube. Avoid employment of pipet or vortex during pellet disruption to maintain cell integrity.
  5. Resuspend cells in 20 mL of complete media and count cells using a neubauer chamber.
  6. Reconstitute cells and dilute in 40 mL of complete media such that 75,000 cells/mL is achieved (2 plates x 96 wells x 200 µL/well = ~40 mL).
  7. Manually mix the cell suspension thoroughly.
  8. Under sterile conditions, dispense 200 µL of the cell suspension per well into a Poly-D-Lysine 96-well microplate (15,000 cells plated per well).
  9. Incubate cells and monitor cell density until 70% confluency is achieved (it takes about 24 hours). Note: 70% confluency is very important. 90 to 100% confluent cells are certain to detach during washing.
  10. Warm 1X PBS and Treatment Buffer to room temperature (RT).
  11. Dissolve the compound of interest in Treatment Buffer.
  12. Remove cell media from plate wells by aspiration.
  13. Wash cells once with 1X PBS (150 µL/well).
  14. Add either Treatment Buffer alone for control cells (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.
  15. Incubate at 37 °C for 30-60 minutes.
  16. Remove activation or stimulation media manually or by aspiration. Immediately (row by row) fix cells with 3.7% Fixing Buffer. 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 cells from the well bottom.
  17. Remove by aspiration Fixing Buffer to an appropriate waste container.
  18. Using a multi-channel pipettor, add 200 µL of 1X PBS. Make sure to carefully add the solution down the sides of the wells to avoid detaching the cells from the well bottom.
  19. Wash five times with 1X PBS for 5 minutes per wash. Allow wash to shake on a rotator for 5 minutes at RT. Always removed wash solution by aspiration. Note: Do not allow cells/wells to become dry during washing. Immediately add the next wash after aspiration.
  20. Using a multi-channel pipettor, block cells/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 cells from the well bottom.
  21. Allow blocking for 90 minutes at RT with moderate shaking on a rotator.
  22. Remove the blocking buffer by aspiration.
  23. Immediately add the primary antibody diluted in 1X PBS (100 µL/well). Do not forget to leave 3 wells for just secondary antibody. Each primary antibody requires a specific dilution. Refer to the antibody manual for the recommended starting dilution.
  24. Incubate with primary antibody overnight at 4°C.
  25. Wash 3 times with 1X PBS for 5 minutes per wash. Allow wash to shake on a rotator for 5 minutes at RT. Using a multi-channel pipettor, add 200 µL/well of 1X PBS. Make sure to carefully add the solution down the sides of the wells to avoid detaching the cells from the well bottom.
  26. Dilute the Alkaline Phosphatase labeled secondary antibody on 1X PBS (use the recommended dilution from the manufacture).
  27. Add secondary antibody diluted on 1X PBS (100 µL/well).
  28. Incubate for 120 minutes with gentle shaking at RT. Protect plate from light during incubation.
  29. Wash 4-5 times (depends on the secondary antibody manufacture) with 1X PBS for 5 minutes per wash. Allow wash to shake 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 cells from the well bottom.
  30. 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 develop buffer (100 µL/well).
  31. Scan 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%.
         

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