Product Description
cAMP and cGMP are small cyclic nucleotides acting as intracellular second messengers. cAMP, produced by adenylyl cyclase and degraded by phosphodiesterase, plays a key role in signal transduction pathways. It is implicated in various physiological and pathological processes, including cardiovascular diseases, cognitive enhancement, and treatment of erectile dysfunction (ED).
This kit uses a competitive enzyme-linked immunoassay (ELISA) to measure cAMP levels in cell extracts or in vitro adenylyl cyclase experiments. The assay involves competition between sample cAMP and HRP-labeled cAMP for binding to an anti-cAMP monoclonal antibody. Absorbance is measured at 450 nm, inversely proportional to cAMP concentration in the sample. The kit features high sensitivity with an IC50 of ~12 pmol/mL and a detection limit of ~0.15 pmol/mL.
Experimental Overview
- Catalog No: ATK00049
- Detection wavelength: 450 nm (reference 630 nm)
- Sensitivity: 0.15 pmol/mL
- IC50: ~12 pmol/mL
Components & Storage
| Component | Details | Storage |
|---|---|---|
| cAMP Standard | 10000 pmol/mL, 1x0.1mL | -80°C |
| cAMP-HRP | 1000x HRP conjugated with cAMP, 1x0.02mL | -80°C |
| cAMP Antibody | Anti-cAMP monoclonal antibody (1000x), 1x0.02mL | -80°C |
| 10x Assay Buffer | Phosphate buffer with preservative, 1x30mL | 2-8°C |
| Neutralizing Reagent | Tris buffered saline with preservative, 1x8mL | 2-8°C |
| Sample Diluent | 0.1M HCl, 1x30mL | 2-8°C |
| TMB Substrate | 3,3’,5,5’-Tetramethylbenzidine, 1x18mL | 2-8°C |
| Stop Solution | 2M sulfuric acid, 1x8mL | 2-8°C |
| Coated Plate | Goat anti-Mouse IgG, 96 wells | 2-8°C |
Procedure Summary
- Prepare standard solutions, samples, and reagents according to the kit instructions.
- Add Neutralizing Buffer, HCl, standards, or samples to the appropriate wells.
- Add cAMP-HRP conjugate and anti-cAMP antibody to each well (excluding controls).
- Incubate at room temperature for 2 hours.
- Wash the plate 4 times with Assay Buffer and remove residual liquid.
- Add TMB Substrate to each well and incubate for 10 minutes.
- Add Stop Solution and measure absorbance at 450 nm.
Storage & Notes
- Store all reagents as instructed in the kit manual.
- Bring all reagents to room temperature before use.
- Avoid cross-contamination by using clean pipette tips and washing thoroughly.
- Handle Stop Solution with care as it is corrosive.
