|Product Includes||Quantity (with Count)||Solution Color|
|cGMP Rabbit mAb Coated Microwells||1 x 96 tests|
|cGMP-HRP Conjugate||1 x 5.5 ml||Red|
|cGMP Standard (5 uM)||1 x 0.5 ml|
|Luminol/Enhancer Solution||1 x 3 ml|
|Stable Peroxide Buffer||1 x 3 ml|
|Sealing Tape||1 x 2 ea|
|ELISA Wash Buffer (20X) 9801||1 x 10 ml|
|Cell Lysis Buffer (10X) 9803||1 x 15 ml|
NOTE: If cell debris is observed it can be removed by brief centrifugation of the plate and transfer of the clear lysates to a new 96 well plate.
Make cGMP standard in the 1X Cell Lysis buffer: Take 50 µl of the cGMP standard (5 µM) and add it to 450 µl diluent to get 500 nM cGMP. Perform a 1:3 serial dilution of this standard to get 160.7 nM, 55.6 nM, 18.5 nM, 6.2 nM, 2.1 nM, 0.7 nM,and 0 nM. The diluent without cGMP will serve as the 0 nM cGMP.
NOTE: The standard curve is used to calculate the absolute amount of cGMP in the sample and is necessary for each assay.
Use a plate-based luminometer to measure Relative Light Units (RLU) at 425 nM within 1-10 minutes following addition of the substrate. Optimal signal intensity is achieved when read within 10 minutes.
Protocol Id: 507
All Species Expected
Cyclic guanosine 3’,5’-monophosphate (cGMP) is a critical and multifunctional second messenger molecule involved in many signal transduction pathways in different cell types of almost all species (1). Intracellular cGMP is generated from GTP by guanylyl cyclase (GC) and degraded through phosphodiesterase (PDE) hydrolysis (1,2). Two distinctive families of GC have been identified: soluble guanylyl cyclases (sGC) that are nitric oxide-responsive and cell membrane-bound; and particulate guanylyl cyclases (pGC) that respond to diverse extracellular agonists including peptide hormones, bacterial toxins, and free radicals (2,3). Phosphodiesterases form a superfamily of 11 isoforms with different specificity to both cyclic adenosine 3’,5’-monophosphate (cAMP) and cGMP (4). Cyclic GMP regulates cellular physiology by activating cGMP-dependent kinase, modulating cGMP-dependent ion channels or transporters, and altering its own hydrolytic degradation by PDE (1,4). Because of the diversity of its effectors, cGMP plays an important role in regulating various pathological and physiological processes, such as vascular smooth muscle motility, intestinal fluid and electrolyte homeostasis, and retinal phototransduction (1,5).
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