| 货号 | 4360S |
| 描述 | 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. |
| 反应种属 | All |
| 应用 | ELISA |
| 目标/特异性 | 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). |
| 供应商 | CST |
| 背景 | 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). |
| 存放说明 | -20C |
| 参考文献 | 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. |
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. 图1：cGMP标准溶液用1X Cell Lysis Buffer #9803稀释，样本处理按照Cyclic GMP XP? 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。 | |
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. 图2：使用硝普钠（SNP）处理RFL-6细胞，Cyclic GMP XP? Assay Kit #4360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。吸光值（左）和活性百分数（右）如上所示。活性百分数按下述计算：% activity=100x[(A-Abasal)/(Amax-Abasal)]，A为样本吸光度，Amax是最大刺激（如高浓度SNP）吸光度，Abasal是基线水平（无SNP）吸光度。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。 | |
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. 图3：使用Forskolin #3828处理RFL-6细胞，Cyclic GMP XP? Assay Kit #4360用来检测心钠素（ANP）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ANP处理30分钟，然后用1X Cell Lysis Buffer #9803裂解。吸光值（左）和活性百分数（右）如上所示。活性百分数按下述计算：% activity=100x[(A-Abasal)/(Amax-Abasal)]，A为样本吸光度，Amax是最大刺激（如高浓度ANP）吸光度，Abasal是基线水平（无ANP）吸光度。ANP是一个结合和激活膜结合的鸟苷酸环化酶的配体，可增加cGMP细胞内浓度。 |
