Screen Quest™ Colorimetric ELISA cAMP Assay Kit

Protocol summary

1. Prepare samples
2. Add 75 µL/well of cAMP standard or test samples into the anti-cAMP coated 96-well plate
3. Incubate at room temperature for 5-10 mins
4. Add 25 µL/well of 1X HRP-cAMP Conjugate
5. Incubate at room temperature for 3 hours
6. Wash 4 times with 200 µL/well Washing Buffer
7. Add 100 µL/well of Amplite™ Green
8. Incubate at room temperature for 1 to 3 hours
9. Monitor absorbance increase at 405, 650 or 740 nm

Important notes
Do not freeze Anti-cAMP Ab Pre-coated 96-well plate (Component F), store it at 4°C. Allow all the kit components to warm to room temperature before using them. Some material might be stick to the vial cap during the shipment. Briefly centrifuge the vial to collect all the content.

1. cAMP stock solution (100 µM):
Add 1 mL of Assay Buffer (Component B) to the vial of cAMP Standard (Component A).

2. HRP-cAMP conjugate stock solution (50X):
Add 55 µL (Cat. # 36370) or 550 µL (Cat. # 36371) of Assay Buffer (Component B) into the vial of HRP-cAMP Conjugate (Component C). Note: The unused 50X HRP-cAMP conjugate stock solution should be divided into single use aliquots and stored them at -20 ^oC.

3. Washing solution (1X):
Add 1 mL of 10X Wash Solution (Component D) to 9 mL distilled water.

HRP-cAMP Conjugate working solution:
Make 1:50 dilution with Assay Buffer (Component B) to have 1X HRP-cAMP conjugate working solution before use. Store it on ice or 4°C. Note: 25 µL of 1X HRP-cAMP conjugate working solution is enough for one assay point; prepare appropriately volume for single use only.

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For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

Prepare samples

1. Treat cells as desired:
   The following is an example of Hela cells treated with Forskolin to induce cAMP in a 96-well plate format: Aspirate off cell growth medium, add 100 µL/well 100 µM Forskolin in Hanks and 20 mM Hepes buffer (HHBS), incubate in a 5% CO₂, 37°C incubator for 15 minutes. Aspirate off cell solution after the incubation, add 100 µL/well of Cell Lysis Buffer (Component E), and incubate at room temperature for another 10 minutes. This cell lysate can be assayed directly or after diluted in Assay Buffer (Component B). Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density. Cells may be seeded the day before or on the day of the experiment depending upon the cell type and/or the effect of the test compounds.
   
   
2. Tissue Samples:
   It is important to rapidly freeze tissues after collection (e.g., using liquid nitrogen) due to quick metabolism of cyclic nucleotides in tissue. Weigh the frozen tissue and add 10 - 20 µL/mg of cell lysis buffer. Homogenize the sample on ice. Spin at top speed for 5 minutes and collect the supernatant. The supernatant may be assayed directly.
   
   
3. Urine, Plasma and Culture Medium Samples:
   Urine and plasma may be tested directly with 1:200 to 1:1000 dilutions in 1X Lysis Buffer. Culture medium can also be tested with 1:10 to 1:200 dilutions in Lysis Buffer. Note: RPMI medium may contain > 350 fmol/µL cAMP.

cAMP assay

1. All the assay wells will be prepared in the following orders: A) cAMP standards, control, or tests samples; B) HRP-cAMP Conjugate.
   
   
2. Add 75 µL/well of the cAMP diluted standard solution and test samples into each well of the anti-cAMP Ab coated 96-well plate (Component F). We recommended duplicating the assays for each standard and testing sample. Incubate at room temperature for 5 to 10 minutes.
   
   
3. Add 25 µL/well of 1X HRP-cAMP Conjugate working solution. Incubate at room temperature for 3 hours by placing the plate on shaker.
   
   
4. Aspirate plate contents, and wash 4 times with 200 µL/well of 1X wash solution.
   
   
5. Add 100 µL/well of Amplite™ Green (Component G) into each well, and incubate at room temperature for 60 mins to 3 hours, protected from light.
   
   
6. Monitor the absorbance increase at 405 nm, 650 nm, or 740 nm using an absorbance plate reader.