Cyclic GMP XP Chemiluminescent Assay Kit是一个竞争性酶联免疫试验，用以分析目标细胞和组织的cGMP水平。此实验中，cGMP被发现在测试样本中与固定数量的HRP连接的cGMP竞争结合固定在96孔板上的抗cGMP XP兔单克隆抗体。接下来的洗涤移除了多余的样本cGMP和HRP连接的cGMP，然后加入Chemiluminescent（化学发光剂）以显色。由于此法的竞争性，使用相对发光单位（RLU）测量的发光强度与样本中cGMP的数量呈反比。使用cGMP标准溶液进行发光值测量，可计算出目标样本中cGMP的绝对数量。

ELISA

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.

图1：cGMP标准溶液用1X Cell Lysis Buffer #9803稀释，样本处理按照Cyclic GMP XP® Chemiluminescent Assay Kit指南操作。此标准曲线仅作为演示使用；使用者应为每一个样本组制作标准曲线，以备精确测定cGMP浓度。

 

ELISA

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.

图2：使用硝普钠（SNP）处理RFL-6细胞，Cyclic GMP XP? Chemiluminescent Assay Kit #8020用来检测硝普钠（SNP）导致的cGMP浓度增加。RFL-6细胞种于2×10^5/孔的12孔板中孵育过夜。细胞分为不处理组，或者0.5 mM IBMX处理15分钟后SNP处理30分钟，然后用1X Cell Lysis Buffer #9803裂解。发光值（左）和活性百分数（右）如上所示。活性百分数按下述计算：% activity=100x[((RLU-RLUbasal)/(RLUmax-RLUbasal)]，RLU为样本发光度，RLUm是最大刺激（如高浓度SNP）发光度，RLUbasal是基线水平（无SNP）发光度。SNP是一个一氧化氮供体，可以直接激活可溶性鸟苷酸环化酶，增加细胞内cGMP浓度。IBMX是非特异性cAMP和cGMP磷酸酶的抑制剂，可以促进cAMP和cGMP在细胞内积累。

 

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; 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).

环鸟苷酸3'-5'-单磷酸酶（cGMP）是一个重要的多功能第二信使，在几乎所有物种不同类型的细胞中涉及许多信号转导通路（1）。细胞内cGMP由鸟苷酸环化酶（GC）催化GTP形成，由磷酸二酯酶（PDE）水解降解（1,2）。GC的两个独特的家族已经确定：可溶性鸟苷酸环化酶（sGC），对一氧化氮反应并和细胞膜结合；颗粒鸟苷酸环化酶（pGC），对不同的细胞外激动剂反应，包括肽类激素，细菌毒素和自由基（2,3）。磷酸二酯酶由11个亚型形成一个超家族，对cAMP和cGMP的特异性各异（4）。cGMP通过激活cGMP依赖的激酶调节细胞生理功能，调节cGMP依赖的离子通道或转运体，通过PDE进行水解降解（1,4）。介于其具有多个效应分子，cGMP在调节多种生理和病理过程中扮演重要角色，例如血管平滑肌运动、肠液和电解质平衡以及视网膜光电转换（1,5）。

Cell细胞增殖/生存能力/健康

细胞死亡

第二信使

活化的小GTPase检测

非放射性的激酶分析试剂盒