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?Cyclic GMP XP Chemiluminescent Assay Kit是一個競爭性酶聯免疫試驗，用以分析目標細胞和組織的cGMP水平。此實驗中，cGMP被發(fā)現在測試樣本中與固定數量的HRP連接的cGMP競爭結合固定在96孔板上的抗cGMP XP兔單克隆抗體。接下來的洗滌移除了多余的樣本cGMP和HRP連接的cGMP，然后加入Chemiluminescent（化學發(fā)光劑）以顯色。由于此法的競爭性，使用相對發(fā)光單位（RLU）測量的發(fā)光強度與樣本中cGMP的數量呈反比。使用cGMP標準溶液進行發(fā)光值測量，可計算出目標樣本中cGMP的絕對數量。

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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.

圖1：cGMP標準溶液用1X Cell Lysis Buffer #9803稀釋，樣本處理按照Cyclic GMP XP? Chemiluminescent Assay Kit指南操作。此標準曲線僅作為演示使用；使用者應為每一個樣本組制作標準曲線，以備精確測定cGMP濃度。

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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.

圖2：使用硝普鈉（SNP）處理RFL-6細胞，Cyclic GMP XP? Chemiluminescent Assay Kit #8020用來檢測硝普鈉（SNP）導致的cGMP濃度增加。RFL-6細胞種于2×10^5/孔的12孔板中孵育過夜。細胞分為不處理組，或者0.5 mM IBMX處理15分鐘后SNP處理30分鐘，然后用1X Cell Lysis Buffer #9803裂解。發(fā)光值（左）和活性百分數（右）如上所示?；钚园俜謹蛋聪率鲇嬎悖? activity=100x[((RLU-RLUbasal)/(RLUmax-RLUbasal)]，RLU為樣本發(fā)光度，RLUm是最大刺激（如高濃度SNP）發(fā)光度，RLUbasal是基線水平（無SNP）發(fā)光度。SNP是一個一氧化氮供體，可以直接激活可溶性鳥苷酸環(huán)化酶，增加細胞內cGMP濃度。IBMX是非特異性cAMP和cGMP磷酸酶的抑制劑，可以促進cAMP和cGMP在細胞內積累。

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Background

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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).

環(huán)鳥苷酸3'-5'-單磷酸酶（cGMP）是一個重要的多功能第二信使，在幾乎所有物種不同類型的細胞中涉及許多信號轉導通路（1）。細胞內cGMP由鳥苷酸環(huán)化酶（GC）催化GTP形成，由磷酸二酯酶（PDE）水解降解（1,2）。GC的兩個獨特的家族已經確定：可溶性鳥苷酸環(huán)化酶（sGC），對一氧化氮反應并和細胞膜結合；顆粒鳥苷酸環(huán)化酶（pGC），對不同的細胞外激動劑反應，包括肽類激素，細菌毒素和自由基（2,3）。磷酸二酯酶由11個亞型形成一個超家族，對cAMP和cGMP的特異性各異（4）。cGMP通過激活cGMP依賴的激酶調節(jié)細胞生理功能，調節(jié)cGMP依賴的離子通道或轉運體，通過PDE進行水解降解（1,4）。介于其具有多個效應分子，cGMP在調節(jié)多種生理和病理過程中扮演重要角色，例如血管平滑肌運動、腸液和電解質平衡以及視網膜光電轉換（1,5）。

Cell細胞增殖/生存能力/健康

細胞死亡

第二信使

活化的小GTPase檢測

非放射性的激酶分析試劑盒