This cell-based assay is based on HTRF® technology (Homogeneous Time-Resolved Fluorescence). It is a competitive immunoassay that uses cAMP labeled with the d2 acceptor flourophore and an anti-cAMP monoclonal AB labeled with Europium Cryptate. The FRET signal decreases as cAMP concentration rises.
Agonist Assay Protocol
1. Seed 5 μl CHO-K1/D1 cells into a 384-well low volume plate, 3,000 cells per well.
2. Add 5 μl compound or dopamine (diluted in buffer) to each well and incubate the plate for 30 min at 23°C.
3. Add 5ul of cAMP-d2 conjugate solution to each well.
4. Add 5μl of cAMP-AB lysis buffer solution to each well.
5. Incubate the plate in the dark for one hour at 23°C.
6. Read the plate PHERAstar PLUS (BMG Labtech, Offenburg, Germany).
CHO-K1/D1 Stable Cell Line
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