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Stock availability
In Stock
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Kit s description
Chemiluminescent 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 CLIA Kits
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Other name
3'-5'-cyclic adenosine monophosphate CLIA Kit
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Brand name
StressXpress®
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Detection system
Chemiluminescent Assay
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Assay format
Direct CLIA (Chemiluminescent Immunoassay)
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Main intended usage
CLIA kit used to quantitatively measure cAMP present in samples.
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Brief protocol
The Cyclic AMP (cAMP) CLIA (High-Sensitivity) 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. This kit can measure as little as 1 femtomol cAMP per sample. 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 white microtiter plate coated with an antibody to capture sheep IgG is provided. Prior to the addition of any samples or standards 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 the chemiluminescent substrate is added. The substrate reacts with the bound cAMP-peroxidase conjugate to produce light The generated light is detected in a microtiter plate reader capable of reading luminescence. 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 White 96 Well Plates, Cyclic AMP Standard, StressXpress® Cyclic AMP Antibody, StressXpress® Cyclic AMP Conjugate Concentrate, Conjugate Diluent, Sample Diluent, Plate Primer, Acetic Anhydride, Triethylamine, Wash Buffer Concentrate, Substrate Solution A, Substrate Solution B, Plate Sealer
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Assay precision
Intra Assay Precision (Regular): Two 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- 11.2 pmol/mL, 3.9% CV Sample 2- 3.6 pmol/mL, 8.3% 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- 1.32 pmol/mL, 8.4% CV Sample 2- 0.39[pmol 10.9% CV Inter Assay Precision (Regular): Two human urine samples were diluted with Sample Diluent and run in duplicates in fourteen assays run over multiple days by four operators. The mean and precision of the calculated cAMP concentrations were: Sample 1- 6.5 pmol/mL, 8.1% CV Sample 2- 2.1 pmol/mL, 13.0% CV Inter Assay Precision (Acetylated): Two human plasma sample were diluted with Sample Diluent, acetylated and run in duplicates in fourteen assays run over multiple days by four operators. The mean and precision of the calculated cAMP concentrations were: Sample 1- 1.02 pmol/mL, 10.1% CV Sample 2- 0.32 pmol/mL, 16.5% 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
38 samples in duplicate
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Assay duration
2 Hours
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Kit s sensitivity
0,119 pmol/ml
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Assay detection limit
Regular: 0,185 - 15 pmol/ml, Acetylated: 0,039 - 5 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 CLIA Kit (High-Sensitivity) (Chemiluminescent Immunoassay) StressXpress® - SKT-208. Assay Type: Direct CLIA. Detection Method: Chemiluminescent Assay. Assay Range: Regular: 0.185 - 15 pmol/ml. Acetylated: 0.039 - 5 pmol/ml. | Acetylated Format Typical Standard Curve for the Cyclic AMP CLIA Kit (High-Sensitivity) (Chemiluminescent Immunoassay) StressXpress® - SKT-208. Assay Type: Direct CLIA. Detection Method: Chemiluminescent Assay. Assay Range: Regular: 0.185 - 15 pmol/ml. Acetylated: 0.039 - 5 pmol/ml. | Linearity was determined by taking two Jurkat cell lysate samples, one with a low cAMP level of 0.2 pmol/mL and one with a higher level of 2.5 pmol/mL, and mixing them in the ratios. The measured concentrations were compared to the expected values based on the ratios used. | Chemical structure of Cyclic AMP (cAMP) Graph of the Regular Format Typical Standard Curve for the Cyclic AMP CLIA Kit (High-Sensitivity) StressXpress - SKT-208 | Graph of the Acetylated Format Typical Standard Curve for the Cyclic AMP CLIA Kit (High-Sensitivity) StressXpress - SKT-208 | Graph of the Non-Acetylated Linearity Recovery for the Cyclic AMP CLIA Kit (High-Sensitivity) StressXpress - SKT-208 | Chemical Structure of Cyclic AMP for the Cyclic AMP CLIA Kit (High-Sensitivity) StressXpress - SKT-208
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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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