Bridge-It® Cyclic AMP (cAMP) all in one Assay Kit, 384-well format

cAMP all in one 122938

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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!

 

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Example of Bridge-It® cAMP all in one Assay for Cells in Suspension

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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

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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:

1. Salvatori AS, Elrick MM, Samson WK, Corbett JA, Yosten GL. Neuronostatin inhibits glucose-stimulated insulin secretion via direct action on the pancreatic α-cell. Am J Physiol Endocrinol Metab. 2014 Jun 1;306(11):E1257-63.

–“Sample pellets were resuspended in appropriate assay buffers, and cAMP was measured by radioimmunoassay (PerkinElmer), BioTrak enzyme immunoassay (Amersham, Piscataway, NJ), or Bridge-It fluorometric assay (Mediomics, St. Louis, MO) according to the manufacturers’ protocols.”

2. Mandal S, Mukhopadhyay S, Bandhopadhyay S, Sen G, Biswas T. 14-Deoxyandrographolide alleviates ethanol-induced hepatosteatosis through stimulation of AMP-activated protein kinase activity in rats. Alcohol. 2014 Mar;48(2):123-32

— “Intracellular cAMP levels in rat hepatocytes and human hepatoma HepG2 cells (10 5 /mL) were measured by using Bridge-It ® cAMP Fluorescence Assay kits (Mediomics, LLC).”

3. Lee YS et al. The fractalkine/CX3CR1 system regulates β cell function and insulin secretion. Cell. 2013 Apr 11;153(2):413-25.

— “Intracellular cAMP level of Min6 cells was measured using Bridge-it cAMP assay kit (Mediomics, USA).”

4. McGraw, DW. A Polymorphic Change in Amino Acid 220 (Ser220Cys) Of The Human ß2- Adrenergic Receptor Encodes Receptors With Different Gs-Coupled Signal Transduction Properties.

— “Cellular production of 125 cAMP was measured using a fluorescent assay kit (Mediomics, MO).”

5. Abukhashim M, Wiebe GJ, Seubert JM. Regulation of forskolin-induced cAMP production by cytochrome P450 epoxygenase metabolites of arachidonic acid in HEK293 cells. Cell Biol Toxicol. 2011 Oct;27(5):321-32.

— “Intracellular cAMP levels were measured by using commercially available kits (Mediomics).”

6. Reszka KJ, Sallans L, Macha S, Brown K, McGraw DW, Kovacic MB, Britigan BE. Airway peroxidases catalyze nitration of the {beta}2-agonist salbutamol and decrease its pharmacological activity. J Pharmacol Exp Ther. 2011 Feb;336(2):440-9.

— “The ability of native and nitrated salbutamol to stimulate cAMP production in airway smooth muscle cells was measured by a fluorescent detection assay using a commercially available kit (Mediomics, St. Louis, MO).”

7. Brent D. Wilson, Masaaki Ii, Kye Won Park, Arminda Suli, Gerhardus A.H. Kock, Lise K. Sorensen, Wonhee Suh, Fréderic Larrieu-Lahargue, Lisa D. Urness, Kirk R. Thomas, Chi-Bin Chien, Douglas W. Losordo, and Dean Y. Li.Netrins Promote Developmental and Therapeutic Angiogenesis. Science. 2006 August 4; 313(5787): 640–644.

— “Two different cAMP measurement systems were utilized (Mediomics and Assay Designs); similar results were obtained with each assessment.”

8. Kalhan R, Thavarajah K, Nlend MC, Nair A, Smith LJ, Sporn PHS. The soy isoflavone genistein blocks transforming growth factor b1‐stimulated lung fibroblast to myofibroblast transformation. Journal of Investigative Medicine: 1 March 2006 – Volume 54 – Issue 2 – p S354.

— “Using an assay in which reductions in fluores- cence intensity are associated with increasing cAMP levels (Mediomics, LLC), we quantified cAMP levels in neurospheres (NS) and differentiated cells (DC) from the human cortical fragile X (M049) and control…”

9. Zhang X, Candas M, Griko NB, Taussig R, Bulla LA Jr. (2006) A mechanism of cell death involving an adenylyl cyclase/PKA signaling pathway is induced by the Cry1Ab toxin of Bacillus thuringiensisProc Natl Acad Sci U S A.103:9897-9902.

— “Involvement of AC/PKA pathway in Cry1Ab toxin-induced cytotoxicity. (A) Stimulation of cAMP production by Cry1Ab toxin. Cells were incubated with isobutyl-1-methylxanthine (0.5 mM) before the addition of Cry1Ab, and cAMP was measured by using the Bridge-It cAMP Assay (Mediomics) in S5 and H5 cells (3 x 104).”

10. Keys JR, Zhou RH, Harris DM, Druckman CA, Eckhart AD. (2005) Vascular smooth muscle overexpression of G protein-coupled receptor kinase 5 elevates blood pressure, which segregates with sex and is dependent on Gi-mediated signalingCirculation. 112:1145-1153.

— “cAMP accumulation was measured by use of a Bridge-It cAMP assay kit according to the manufacturer’s instructions (Mediomics, LLC).”


This product is protected by patents and pending patents.

Catalog #: 122939 (100 measurements- one time trial size),122938 (384 measurements)

Protocol: 

SDS: