cAMP PharmaGenie ELISA Kit (SBRS0012)

1. Pre-Coated 96-well Strip Microplate
2. Wash Buffer
3. Standard Peptide
4. Assay Diluent(s)
5. Biotinylated Peptide
6. HRP-Streptavidin
7. TMB One-Step Substrate
8. Stop Solution
9. Assay Diagram
10. Positive Control Sample
11. Capture Antibody
12. Technical Manual

Other materials and equipment required:

The Assay Genie cAMP PharmaGenie ELISA Kit (SBRS0012) will require other equipment and materials to carry out the assay. Please see list below for further details.

• Distilled or deionized water
• Precision pipettes to deliver 2 ul to 1 ml volumes
• Adjustable 1-25 ml pipettes for reagent preparation
• 100 ml and 1 liter graduated cylinders
• Tubes to prepare standard and sample dilutions
• Orbital shaker
• Aluminum foil
• Saran Wrap
• Absorbent paper
• Microplate reader capable of measuring absorbance at 450nm
• SigmaPlot software (or other software that can perform four-parameter logistic regression models)

1. Prepare all reagents, samples and standards as instructed.
2. Add 100 ul detection antibody to each well.
3. Incubate 1.5 h at RT or O/N at 4°C.
4. Add 100 ul standard or sample to each well.
5. Incubate 2.5 h at RT.
6. Add 100 ul prepared streptavidin solution.
7. Incubate 45 min at RT.
8. Add 100 ul TMB One-Step Substrate Reagent to each well.
9. Incubate 30 min at RT.
10. Add 50 ul Stop Solution to each well.
11. Read plate at 450 nm immediately.

Cyclic adenosine monophosphate (cAMP, cyclic AMP, or 3',5'-cyclic adenosine monophosphate) is a second messenger important in many biological processes. cAMP is derived from adenosine triphosphate (ATP) and used for intracellular signal transduction in many different organisms, such as transferring into cells the effects of hormones like glucagon and adrenaline, which cannot pass through the plasma membrane. cAMP functions through three main effectors: PKA, the guanine-nucleotide-exchange factor (GEF) EPAC and cyclic-nucleotide-gated ion channels. The intracellular levels of cAMP are regulated by the balance between the activities of two enzymes adenylyl cyclase (AC) and cyclic nucleotide phosphodiesterase (PDE). Cyclic AMP has been shown to be involved in cell growth, differentiation and general metabolism, and it is important for many biological function, especially in the cardiovascular, nervous and immune systems. The measurement of intracellular Cyclic AMP in tissues and cell cultures may help to provide a clearer understanding of the physiology and pathology of many disease states.