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Stock availability
In Stock
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Kit s description
Quantitative colorimetric detection of cAMP
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Protein target
Cyclic AMP
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Scientific context
Adenosine-3’,5’-cyclic monophosphate (cyclic AMP) is a second messenger that plays an important role in intracellular regulation. Specifically, it functions as a mediator of activity of several key hormones including epinephrine, glucagon, and ACTH (1-4). Adenylate cyclase is activated by the hormones glucagon and adrenaline and by G protein. Liver adenylate cyclase responds more strongly to glucagon, and muscle adenylate cyclase responds more strongly to adrenaline. cAMP decomposition into AMP is catalyzed by the enzyme phosphodiesterase. In the Human Metabolome Database there are 166 metabolic enzymes listed that convert cAMP (5). Other biological actions of cAMP include regulation of innate immune functioning (6), axon regeneration (7), cancer (8), and inflammation (9).
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Primary research area
Cell Signaling, Neuroscience
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Category
Assay EIA Kits
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Other name
3'-5'-cyclic adenosine monophosphate EIA Kit
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Brand name
StressXpress®
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Detection system
Colorimetric Assay
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Assay format
Direct EIA (Enzyme Immunoassay)
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Main intended usage
EIA kit used to quantitatively measure cAMP present in samples.
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Brief protocol
The Cyclic AMP EIA kit is designed to quantitatively measure cAMP present in lysed cells, EDTA and heparin plasma, urine, saliva and tissue culture media samples. For tissue samples, saliva and urine, where the levels of cAMP are expected to be relatively high, the regular format for the assay can be used. For plasma samples and some dilute cell lysates an optional acetylation protocol can be used. The kit is unique in that all samples and standards are diluted into an acidic Sample Diluent, which contains special additives and stabilizers, for cAMP measurement. This allows plasma, urine and saliva samples to be read in an identical manner to lysed cells. Acidified samples of cAMP are stable and endogenous phosphodiesterases are inactivated in the Sample Diluent. A cAMP standard is provided to generate a standard curve for the assay and all samples should be read off the standard curve. A clear microtiter plate coated with an antibody to capture sheep IgG is provided and a neutralizing Plate Primer solution is added to all the used wells. Standards or diluted samples, either with or without acetylation, are pipetted into the primed wells. A cAMP-peroxidase conjugate is added to the standards and samples in the wells. The binding reaction is initiated by the addition of a sheep antibody to cAMP to each well. After a 2 hour incubation, the plate is washed and substrate is added. The substrate reacts with the bound cAMP-peroxidase conjugate. After a short incubation, the reaction is stopped and the intensity of the generated color is detected in a microtiter plate reader capable of measuring 450 nm wavelength. The concentration of the cAMP in the sample is calculated, after making suitable correction for the dilution of the sample, using software available with most plate readers.
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Kit contents
Coated Clear 96 Well Plates, Cyclic AMP Standard, StressXpress® Cyclic AMP Antibody, StressXpress® Cyclic AMP Conjugate, Sample Diluent, Plate Primer, Acetic Anhydride, Triethylamine, Wash Buffer Concentrate, TMB Substrate, Stop Solution, Plate Sealer
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Assay precision
Intra Assay Precision (Regular): Three human urine samples were diluted with Sample Diluent and run in replicates of 20 in an assay. The mean and precision of the calculated cAMP concentrations were: Sample 1- 56.9 pmol/mL, 8.6% CV Sample 2- 11.9 pmol/mL, 11.3% CV Sample 3- 6.7 pmol/mL, 12.3% CV Intra Assay Precision (Acetylated): Two human plasma samples were diluted with Sample Diluent, acetylated and run in replicates of 20 in an assay. The mean and precision of the calculated cAMP concentrations were: Sample 1- 1.74 pmol/mL, 10.4% CV Sample 2- 0.60 pmol/mL, 11.8% CV Inter Assay Precision (Regular): Three human urine samples were diluted with Sample Diluent and run in duplicates in twelve assays run over multiple days by four operators. The mean and precision of the calculated cAMP concentrations were: Sample 1- 57.1 pmol/mL, 10.0% CV Sample 2- 10.9 pmol/mL, 11.5% CV Sample 3- 6.3 pmol/mL, 11.3% CV Inter Assay Precision (Acetylated): One human urine and two human plasma sample were diluted with Sample Diluent, acetylated and run in duplicates in twelve assays run over multiple days by four operators. The mean and precision of the calculated cAMP concentrations were: Sample 1- 5.22 pmol/mL, 9.8% CV Sample 2- 0.99 pmol/mL, 8.1% CV Sample 3- 0.73 pmol/mL, 15.4% CV
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Assay cross reactivity
Species Independent
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Samples to be used with this kit
Cell Lysates, Tissue, Saliva, Urine, EDTA Plasma, Heparin Plasma, Tissue Culture Media
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Maximum samples to be used with this kit
39 samples in duplicate
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Assay duration
2,5 Hours
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Kit s sensitivity
0,64 pmol/ml
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Assay detection limit
Regular: 0,617 - 150 pmol/ml, Acetylated: 0,156 - 10 pmol/ml
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Storage recommendations
4°C
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Shipping recommendations
Blue Ice
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Bibliography
1. Sutherland, E. W. and Rall, T. W. Fractionation and Characterization of a Cyclic Adenine Ribonucleotide Formed by Tissue Particles. J. Biol. Chem., 232:1077, 1958. 2. Marsh, J.M. Biol. Reprod., 14:30-53, 1976. 3. Korenman, S.G. and Krall, J.F. Biol. Reprod., 16:1-17, 1977. 4. Kelley, D.J., Bhattacharyya, A., Lahvis, G.P., Yin, J.C.P., Malter, J., and Davidson, R.J. Neurosci. Biobehav. Rev., 32(8): 1533-1543, 2008. 5. more 6. Serezani, C.H., Ballinger, M.N., Aronoff, D.M., and Peters-Golden, M. Am. J. Resp. Cell and Mol. Biol., 39 (2): 127, 2008. 7. Hannila, S.S., and Filbin, M.T. Exp. Neurol., 209(2): 321–332, 2008. 8. Shankar, D.B, Cheng, J.C., and Sakamoto, K.M. Cancer, 104(9):1819-24, 2005. 9. Galea E. and Feinstein, D.L. FASEB J., 13:2125-2137, 1999.
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Release date
17-Sep-2014
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PubMed number
Refer to PubMed
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Tested applications
No data available / currently testing
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Tested reactivity
No data available / currently testing
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Resources available upon request
Kit Booklet, MSDS, Saliva Sample Handling Instructions
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Product image link
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Product image legend
Regular Format Typical Standard Curve for the Cyclic AMP EIA Kit (Enzyme Immunoassay) StressXpress® - SKT-209. Assay Type: Direct EIA. Detection Method: Colorimetric Assay. Assay Range: Regular: 0.617 - 150 pmol/ml. Acetylated: 0.156 - 10 pmol/ml. | Acetylated Format Typical Standard Curve for the Cyclic AMP EIA Kit (Enzyme Immunoassay) StressXpress® - SKT-209. Assay Type: Direct EIA. Detection Method: Colorimetric Assay. Assay Range: Regular: 0.617 - 150 pmol/ml. Acetylated: 0.156 - 10 pmol/ml. | Chemical structure of Cyclic AMP (Adenosine-3’,5’-cyclic monophosphate) Graph of the Regular Format Typical Standard Curve for the Cyclic AMP EIA Kit StressXpress - SKT-209 | Graph of the Acetylated Format Typical Standard Curve for the Cyclic AMP EIA Kit StressXpress - SKT-209 | Chemical Structure of Cyclic AMP for the Cyclic AMP EIA Kit StressXpress - SKT-209
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Warnings
Non-hazardous
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Country of production
Canada
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Total weight kg
2
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Net weight g
300
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Description
This ELISA test kit or EIA Enzyme immuno assay is an enzyme linked immuno assay supplied as coated 96 well plates with antigen or antibody and needs to be stored at +4°C.
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Properties
MeSH E05 478 566 350 170 Enzyme-Linked Immunosorbent Assay
