Figure 2: Treatment of RFL-6 cells with sodium nitroprusside (SNP) increases cGMP concentration as detected by Cyclic GMP XP® Assay Kit #4360. 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 absorbance values (left) and percentage of activity (right) are shown above. The percentage of activity is calculated as follows: % activity=100x[(A-Abasal)/(Amax-Abasal)], where A is the sample absorbance, Amax is the absorbance at maximum stimulation (i.e., high SNP concentration), and Abasal is the absorbance 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.
Figure 3: Treatment of RFL-6 cells with atrial natriuretic peptide (ANP) increases cGMP concentration as detected by Cyclic GMP XP® Assay Kit #4360. 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 ANP treatment (30 minutes) and lysed with 1X Cell Lysis Buffer #9803. The absorbance values (left) and percentage of activity (right) are shown above. The percentage of activity is calculated as follows: % activity=100x[(A-Abasal)/(Amax-Abasal)], where A is the sample absorbance, Amax is the absorbance at maximum stimulation (i.e., high ANP concentration), and Abasal is the absorbance at basal level (no ANP). ANP is a ligand that binds and activates membrane bound guanylyl cyclases to increase cellular cGMP concentration.
Figure 1: cGMP Standard was diluted in 1X Cell Lysis Buffer #9803 and samples were assayed following the Cyclic GMP XP® 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.
|Product Includes||Quantity (with Count)||Solution Color|
|cGMP Rabbit mAb Coated Microwells||1 x 96 tests|
|cGMP-HRP Conjugate||1 x 11 ml||Red|
|cGMP Standard (5 uM)||1 x 1 ml|
|TMB Substrate 7004||1 x 11 ml|
|STOP Solution 7002||1 x 11 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: This procedure is for a 96-well tissue culture plate. It can be modified for other tissue culture plates (6, 12, 24, or 48 well).
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.
NOTE: Watch the color as it being developed since it may be necessary to stop the reaction before 30 minutes.
NOTE: To determine the absolute amount of the tested substance, a standard curve needs to be generated each time. Please follow the detailed protocol on the product datasheet to determine the concentration range for the standard curve.
posted April 2010
Protocol Id: 68
The Cyclic GMP XP® 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 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, HRP substrate TMB is added to develop color. Because of the competitive nature of this assay, the magnitude of the absorbance for this developed color is inversely proportional to the quantity of sample cGMP. Measurement of absorbance using the cGMP Standard allows calculating the absolute amount of cGMP in a sample of interest.
Note: 12 8-well modules - Each module is designed to break apart for 8 tests.
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 and Figure 3 (low passage RFL-6 cells).
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, 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 phosphodiesterase (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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