Revision 10

#4360Store at -20C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
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Product Information

Storage

Please keep all kit components in -20°C before use.

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).

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.

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).

  1. Domek-Łopacińska, K. and Strosznajder, J.B. (2005) J Physiol Pharmacol 56 Suppl 2, 15-34.
  2. Lucas, K.A. et al. (2000) Pharmacol Rev 52, 375-414.
  3. Potter, L.R. et al. (2006) Endocr Rev 27, 47-72.
  4. Matsumoto, T. et al. (2003) J Smooth Muscle Res 39, 67-86.
  5. Rybalkin, S.D. et al. (2003) Circ Res 93, 280-91.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

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 cellsignal.com/trademarks for more information.

限制使用

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Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
仅供研究使用。不得用于诊断流程。
Revision 10
#4360

Cyclic GMP XP® Assay Kit

Cyclic GMP XP® Assay Kit: Image 1 Expand Image
图 1：cGMP 标准液用 1X Cell Lysis Buffer #9803 稀释，并且样品遵照 Cyclic GMP XP® Assay Kit 实验步骤进行检测。该标准曲线仅用作演示目的；用户应为每个样品组（的测定）建立标准曲线，以准确确定 cGMP 浓度。
Cyclic GMP XP® Assay Kit: Image 2 Expand Image
图 2：RFL-6 细胞用硝普钠 (SNP) 处理会导致 cGMP 浓度升高，使用 Cyclic GMP XP® Assay Kit #4360 可进行检测。RFL-6 细胞按 2x105 个细胞/孔放在 12 孔板上进行培养，并孵育过夜。细胞不用处理或用 0.5 mM IBMX 预处理 15 分钟，随后进行 SNP 处理（30 分钟）并用 1X Cell Lysis Buffer #9803 进行裂解。吸光度值（左图）和活性百分比（右图）如上所示。活动百分比计算如下：% 活性 = 100x [(A-A本底)/(A最大-A本底)]，其中 A 为样品吸光度，A最大为最大刺激（即高 SNP 浓度）时的吸光度，A 本底为在本底水平（无 SNP）时的吸光度。SNP 是一种一氧化氮供体，能直接激活可溶性鸟苷酸环化酶并使细胞 cGMP 浓度升高。IBMX 是 cAMP 和 cGMP 磷酸二酯酶的一种非特异性抑制剂，能促进 cAMP 和 cGMP 在细胞中聚集。
Cyclic GMP XP® Assay Kit: Image 3 Expand Image
图 3：RFL-6 细胞用房钠肽 (ANP) 处理会导致 cGMP 浓度升高，使用 Cyclic GMP XP® Assay Kit #4360 可进行检测。RFL-6 细胞按 2x105 个细胞/孔放在 12 孔板上进行培养，并孵育过夜。细胞未经处理或用 0.5 mM IBMX 预处理 15 分钟，随后进行 ANP 处理（30 分钟）并用 1X Cell Lysis Buffer #9803 进行裂解。吸光度值（左图）和活性百分比（右图）如上所示。活性百分比计算如下：% 活性 = 100x [(A-A本底)/(A最大-A本底)]，其中 A 为样品吸光度，A最大为最大刺激（即高 ANP 浓度）时的吸光度，A 本底为在本底水平（无 ANP）时的吸光度。ANP 是一种配体，能结合并激活膜结合的鸟苷酸环化酶，从而使细胞 cGMP 浓度升高。
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Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
仅供研究使用。不得用于诊断流程。