Mouse/Rat cAMP ELISA Kit

Sample

Cell culture supernates, cell lysates, tissue homogenates, plasma, and urine

Species Reactivity

Mouse; Rat

Detection Method

cELISA

Intended Use

For the quantitative determination of cyclic AMP (cAMP) concentrations in mouse or rat cell culture supernates, cell lysates, tissue homogenates, plasma, and urine.

Contents of Kit

1. Streptavidin Coated Microplate: 96 well polystyrene microplate (12 strips of 8 wells) coated with streptavidin.
Note: Return unused wells to the foil pouch containing the desiccant pack. Reseal along entire edge of the zip-seal.
2. Mouse/Rat cAMP Conjugate: 6 mL of cAMP conjugated to horseradish peroxidase with preservatives.
3. cAMP Standard: cAMP in a buffer with preservatives; lyophilized. Refer to the vial label for reconstitution volume.
4. Biotinylated Primary Antibody: 6 mL of a biotinylated mouse monoclonal antibody to cAMP in buffer with preservatives.
5. Calibrator Diluent: 2 vials (21 mL/vial) of a buffered protein base with preservatives.
6. Cell Lysis Buffer 5: 21 mL/vial of a concentrated buffered solution with preservatives. Use diluted 1:5 in this assay. May contain crystals. Warm to room temperature and mix well to dissolve. May turn yellow over time.
7. Wash Buffer Concentrate: 2 vials (21 mL/vial) of a 25-fold concentrated solution of buffered surfactant with preservative. May turn yellow over time.
8. Color Reagent A: 12 mL of stabilized hydrogen peroxide.
9. Color Reagent B: 12 mL of stabilized chromogen (tetramethylbenzidine).
10. Stop Solution: 23 mL of diluted hydrochloric acid.
11. Plate Sealers: 4 adhesive strips.

Storage

Store unopened kit at ≤ -20 °C in a manual defrost freezer and at 2-8 °C for up to 1 month. Do not use past kit expiration date.

Precision

Intra-Assay Precision (Precision within an assay)
Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision.
Inter-Assay Precision (Precision between assays)
Three samples of known concentration were tested in twenty separate assays to assess inter-assay precision. Assays were performed by at least three technicians using two lots of components.

Sensitivity

Eighty assays were evaluated and the minimum detectable dose (MDD) of mouse/rat cAMP ranged from 0.01-1.54 pmol/mL. The mean MDD was 0.79 pmol/mL.
The MDD was determined by subtracting two standard deviations from the mean O.D. value of twenty zero standard replicates and calculating the corresponding concentration.

General Description

Adenosine 3'5'-cyclic monophosphate (cAMP) is a ubiquitous second messenger that mediates a diverse range of cellular processes in all organisms from bacteria to higher eukaryotes. It is converted from adenosine triphosphate (ATP) by adenylyl cyclases (ACs), and is inactivated by phosphodiesterases (PDEs) which catalyze its hydrolysis to 5'-AMP. In mammals, upon interaction with extracellular ligands, G protein coupled receptors (GPCRs) linked to Gαs activate the family of nine transmembrane ACs to increase intracellular cAMP. In contrast, GPCRs associated with Gαi/o inhibit the synthesis of cAMP by transmembrane ACs. With the exception of AC9, transmembrane ACs can be activated by forskolin, a plant diterpene commonly used to raise cAMP levels in cells. A divalent cation-dependent soluble AC (sAC) isoform also exists. It is activated by bicarbonate ions and can be found in the cytosol and in subcellular organelles.
The physiological roles of cAMP are mediated via multiple effector molecules. Binding of cAMP to protein kinase A (PKA) holoenzyme induces conformational changes and releases the catalytic subunit to phosphorylate target substrates on serine/threonine residues. cAMP binding to the guanine nucleotide exchange factors Epac1/2 (exchange protein activated by cAMP, also known as cAMP-GEF-I and cAMP-GEF-II) mediates the exchange of GDP for GTP on the small molecular weight G proteins Rap-1 and -2. Activated Rap proteins are important in multiple cellular processes including adhesion and exocytosis. cAMP can also activate cyclic nucleotide-gated ion channels (CNG) by binding directly to the nonselective cation channel proteins that are expressed in various tissues. cAMP signaling is spatially and temporally regulated, allowing for the selective activation of a subset of targets. A-kinase anchoring proteins (AKAPs) provide the platform for the assembly of signalsomes consisting of cAMP effectors (PKA and/or Epac) and their substrates, together with signal terminators including phosphatases and PDEs.
In response to increases in intracellular cAMP, a wide variety of cell types possess mechanisms for exporting cAMP to the extracellular space. In mammals, plasma and urine cAMP concentrations can become highly elevated under certain physiological conditions. Extracellular cAMP is known to exert physiological actions on diverse cell types, in part through the cAMP-adenosine pathway where cAMP is converted to adenosine via ecto-PDEs and ectonucleotidases.

Standard Curve

This standard curve is provided for demonstration only. A standard curve should be generated for each set of samples assayed.