Mouse/Rat cAMP Parameter Assay Kit

Assay Procedure

1.   Prepare all reagents, standard dilutions, and samples as directed in the product insert.
2.   Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.
3.   Aspirate each well of the Streptavidin-coated microplate and wash, repeating the process twice for a total of 3 washes.

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50 µL Biotinylated Primary Antibody Solution

4.   Add 50 µL of Biotinylated Primary Antibody Solution to all wells except the non-specific binding (NSB) wells. Cover with a plate sealer, and incubate at room temperature for 1 hour on a horizontal orbital microplate shaker.
5.   Aspirate and wash 4 times.

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50 µL Conjugate

6.   Immediately add 50 µL of Conjugate to each well.

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100 µL Standard, Control, or Sample

7.   Add 100 µL of Standard, Control, or sample to the appropriate wells.

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100 µL of Calibrator Diluent

8.   Add 100 µL of the Calibrator Diluent to the NSB and zero standard (B₀) wells. Cover with a new plate sealer, and incubate at room temperature for 2 hours on the shaker.
9.   Aspirate and wash 4 times.

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100 µL Substrate Solution

10. Add 100 µL Substrate Solution to each well. Incubate at room temperature for 30 minutes on the benchtop. PROTECT FROM LIGHT.

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100 µL Stop Solution

11. Add 100 µL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.

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