Hi all,
I am using the cyclic AMP competitive ELISA kit from Thermo fisher and have been not getting a signal from either my standards or samples. I observe the green color change after adding the yellow cAMP antibody indicating that the wells have been pre-coated. But after washing and adding substrate solution none of my samples or standards develop.
I would greatly appreciate any advice from anyone with technical knowledge of cAMP ELISA kits.
ThanksViji,
The only issue is that not only the samples but also the cAMP standard provided in the kit do not show a signal.
I see a technical problem there. Probably more related to measurement than to incubations but you may check both.
You may consider to move to other more "reliable" methods (at least for us), namely HTRF based. I copy our methodology as written in one of our papers:
Forskolin dose-response curves in different density of cells were performed to select the most appropriate conditions of the assay, which resulted in 5,000 HEK-293T cells, 7,500 neurons and 0.5 µM forskolin. Subsequently, assays were performed in medium containing 50 μM zardeverine, placing cells in 384-well microplates. This was done by the preincubation with reagents ... , followed by .... addition (100 nM final concentration) and, after 15 min incubation period 50 µM forskolin was added. Readings were performed 15 min later using a homogeneous time-resolved fluorescence energy transfer (HTRF) method requiring the Lance Ultra cAMP kit (PerkinElmer) and fluorescence readings (at 665 nm) in a PHERAstar Flagship microplate reader
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