Bridge-It® Cyclic AMP (cAMP) all in one Assay Kit, 384-well format
The Bridge-It® cAMP assay is highly specific assay to quantify cAMP in stimulated cells. ATP, AMP, and cGMP have all been tested for selectivity using the cAMP assay. No response was detected using the Bridge-It® cAMP assay with any of these compounds within the concentration range that is expected in biological samples (i.e., millimolar range for AMP and ATP, and micromolar range for cGMP).
An all-in-one solution is added to activated cells and incubated for 30 minutes. Clarified lysate samples are transferred to a black 384-well microplate to read the fluorescence signal.
$175.00 – $495.00
Description
Bridge-It® Cyclic AMP (cAMP) Assays – Selectivity, Sensitivity and Performance Characteristics
The Bridge-It® Cyclic AMP (cAMP) assay is highly specific. ATP, AMP, and cGMP have all been tested for selectivity using the cAMP assay. No response was detected using the Bridge-It® cAMP assay with any of these compounds within the concentration range that would be expected to occur in “real life” samples (i.e., millimolar range for AMP and ATP, and micromolar range for cGMP). The sensitivity and performance characteristics of the Bridge-It®cAMP designer and the Bridge-It® all in one assay products are presented in the following Table and Figures.
Bridge-It® cAMP Assay |
Microplate Format |
cAMP Detection Level |
designer |
96-well |
5nM (0.5 pmol/well in 100 µl vol.) |
all in one |
384-well |
5nM (100 fmol/well in 20 µl vol) |
Pricing: The 100-well kit is a one-time trial purchase. For bulk pricing options, please contact us. For any other questions or comments, please do not hesitate to contact us. We are always happy to help!
Example of Bridge-It® cAMP all in one Assay for Cells in Suspension
The Bridge-It® cAMP all in one assay allows for the stimulation and measurement of cAMP in adherent monolayer cells attached to the wells of a tissue culture microplate. In the following example, HEK 293 cells were trypsinized and plated into the wells of a 96-well polystyrene tissue culture microplate at a density of 25,000 cells per well in 50 µl of culture media. The cells were allowed to attach to the bottom of the wells overnight. The media was removed the next day and the wells were washed with a buffered saline solution. The wash buffer was then removed and replaced with 50 µl of KRB-IBMX buffer. For stimulation, 1 µl of forskolin at different concentrations was added to each well and incubated and rotated gently for 15 minutes at ~25ºC. To quantify cAMP in each well, the forskolin buffer solution was removed, 100 µl of the all in one Assay Solution was added to each well, and the microplate was incubated for 30 minutes at ~25ºC with rotation. The well contents were then transferred into a black 96-well microplate and the fluorescent signal was read using the settings for fluorescein (~485 nm excitation, ~540 nm emission).
Example of Bridge-It® cAMP all in one Assay for Attached Cells
For information on the assay principle please see the Bridge-It Assay Technology platform page or download the protocol.
This product is protected by patents and pending patents.
Selected Customer Publications using Mediomics’ Bridge-It cAMP Fluorescence Assay Kit:
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Neuronostatin inhibits glucose-stimulated insulin secretion via direct action on the pancreatic α-cell. Salvatori AS, Elrick MM, Samson WK, Corbett JA, Yosten GL. Am J Physiol Endocrinol Metab. 2014 Jun 1;306(11):E1257-63.
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14-Deoxyandrographolide alleviates ethanol-induced hepatosteatosis through stimulation of AMP-activated protein kinase activity in rats. Mandal S, Mukhopadhyay S, Bandhopadhyay S, Sen G, Biswas T. Alcohol. 2014 Mar;48(2):123-32.
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The fractalkine/CX3CR1 system regulates β cell function and insulin secretion. Lee YS, Morinaga H, Kim JJ, Lagakos W, Taylor S, Keshwani M, Perkins G, Dong H, Kayali AG, Sweet IR, Olefsky J. Cell. 2013 Apr 11;153(2):413-25.
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Regulation of forskolin-induced cAMP production by cytochrome P450 epoxygenase metabolites of arachidonic acid in HEK293 cells. Abukhashim M, Wiebe GJ, Seubert JM. Cell Biol Toxicol. 2011 Oct;27(5):321-32.
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Netrins promote developmental and therapeutic angiogenesis. Wilson BD, Ii M, Park KW, Suli A, Sorensen LK, Larrieu-Lahargue F, Urness LD, Suh W, Asai J, Kock GA, Thorne T, Silver M, Thomas KR, Chien CB, Losordo DW, Li DY. Science. 2006 Aug 4;313(5787):640-4.
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A mechanism of cell death involving an adenylyl cyclase/PKA signaling pathway is induced by the Cry1Ab toxin of Bacillus thuringiensis. Zhang X, Candas M, Griko NB, Taussig R, Bulla LA Jr. Proc Natl Acad Sci USA. 2006 Jun 27;103(26):9897-902
