View Featured Offers >>
4360
Cyclic GMP XP® Assay Kit
Cellular Assay Kits
Assay Kit

Cyclic GMP XP® Assay Kit #4360

Citations (0)
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.
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.
To Purchase # 4360
Cat. # Size Qty. Price
4360S
1 Kit  (96 assays)

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

Product Description

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.

Specificity / Sensitivity

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).

Background

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).

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

For Research Use Only. Not for Use in Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit our Trademark Information page.