Figure 1: cGMP Standard was diluted in 1X Cell Lysis Buffer #9803 and samples were assayed following the Cyclic GMP XP® Chemiluminescent Assay Kit protocol. This standard curve is for demonstration purposes only; users should generate a standard curve for each sample set in order to accurately determine cGMP concentration.
Figure 2: Treatment of RFL-6 cells with sodium nitroprusside (SNP) increases cGMP concentration as detected by Cyclic GMP XP® Chemiluminescent Assay Kit #8020. RFL-6 cells were seeded at 2x105 cells/well in a 12-well plate and incubated overnight. Cells were either left untreated or pretreated with 0.5 mM IBMX for 15 minutes prior to SNP treatment (30 minutes) and lysed with 1X Cell Lysis Buffer #9803. The light emission values (left) and percentage of activity (right) are shown above. The percentage of activity is calculated as follows: % activity=100x[(RLU-RLUbasal)/(RLUmax-RLUbasal)], where RLU is the relative light unit, RLUmax is the light emission at maximum stimulation (i.e., high SNP concentration), and RLUbasal is the light emission at basal level (no SNP). SNP is a nitric oxide donor that directly activates soluble guanylyl cyclases and increases cellular cGMP concentration. IBMX is a non-specific inhibitor of cAMP and cGMP phosphodiesterases that promotes accumulation of cAMP and cGMP in cells.
|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
The Cyclic GMP XP® Chemiluminescent Assay Kit is a competition enzyme-linked immunoassay used to determine cGMP levels in cells or tissues of interest. In this assay, cGMP found in the test sample competes with a fixed amount of HRP-linked cGMP for binding to an anti-cGMP XP® Rabbit mAb immobilized onto a 96-well plate. Following washing to remove excess sample cGMP and HRP-linked cGMP, chemiluminescent reagent is added for signal development. Because of the competitive nature of this assay, the magnitude of light emission, measured in relative light units (RLU), is inversely proportional to the quantity of sample cGMP. Measurement of light emmision using the cGMP Standard allows calculating the absolute amount of cGMP in a sample of interest.
The immunoreactivity of this kit was tested against the following: ADP, AMP, ATP, cAMP, cGMP, cIMP, cTMP, CTP, GDP, GMP, and GTP. Minor cross-reactivity was observed with cIMP, with over 100-fold higher sensitivity for cGMP compared to cIMP. No cross-reactivity was observed with any of the other factors tested. Kit sensitivity, as shown in Figure 1, demonstrates a dynamic range of 2 to 200 nM of cGMP. Changes in cellular cGMP levels following specific treatments are shown in Figure 2 (low passage RFL-6 cells).
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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