葡萄糖-6-磷酸脱氢酶 (G6PDH) 活性检测试剂盒（紫外微量法）

产品介绍

  葡萄糖-6-磷酸脱氢酶（G6PD 或 G6PDH）（EC 1.1.1.49）是一种催化化学反应的胞质酶。该酶参与戊糖磷酸途径，这是一种通过维持 NADPH 水平，向细胞（如红细胞）提供还原能量的代谢途径。NADPH 反过来维持这些细胞中谷胱甘肽的水平，保护红细胞免受过氧化氢等化合物的氧化损伤。  检测原理：样本中存在的G6PDH将NADP⁺转化为NADPH，NADPH 在340nm处有特征吸收峰。NADPH 的吸光度值与样本中存在的G6PDH活性成正比。  适用样本：血清（浆）、动植物组织、细胞、细菌G1506775Component48T96TStorageG1506775AAssay Buffer70 mL70 mL×22-8℃G1506775B6-Phosphogluconic Acid1EA1EA2-8℃. Store in the dark.G1506775CNADP⁺1EA1EA2-8℃. Store in the dark.注意：正式检测前，建议选择2-3个预期差异较大的样本进行预实验。自备仪器和试剂1、酶标仪或紫外分光光度计（能测 340nm处的吸光度） 2、96孔UV微孔板或微量石英比色皿、可调节式移液枪及枪头3、去离子水4、匀浆器（如果是组织样本）实验流程1、试剂准备试剂名称试剂准备注意事项Assay Buffer即用型；使用前平衡到室温4℃保存6-Phosphogluconic Acid Working Regent临用前，用2.4mL Assay Buffer溶解管内组分，混合均匀。使用时，请置于冰上。配制好4℃避光可保存1周NADP⁺ Working Regent临用前，用2.4mL Assay Buffer溶解管内组分，混合均匀。使用时，请置于冰上。配制好4℃避光可保存1周2、样本制备2.1 动物组织称取约0.1g样本，加入1mL Assay Buffer，冰浴匀浆，8,000g，4℃离心10min，取上清液，置冰上待测。2.2 植物组织称取约0.1g样本，加入1mL Assay Buffer捣碎，冰浴匀浆，8,000 g，4℃离心10min，取上清液，置冰上待测。2.3 细胞或细菌收集2,000万细胞或细菌到离心管内，用冷PBS清洗细胞，12,000g，4℃离心1min弃上清，加入1mL Assay Buffer，冰浴超声波破碎细胞或细菌5min（功率20%或200W，超声3s，间隔7s，重复30次），8,000g，4℃离心10min，取上清液，置冰上待测。 3.4 血清（浆）等液体样本直接测定。 注意：如需测定蛋白浓度，推荐使用阿拉丁B665595 BCA蛋白定量试剂盒或R1491648 即用型BCA蛋白定量试剂盒。3、实验步骤3.1 酶标仪或紫外分光光度计预热30min以上，调节波长到340nm，紫外分光光度计用去离子水调零。3.2 Assay Buffer置于25℃或者37℃水浴预热30min以上。 3.3 取96孔UV板，按如下方式进行加样：试剂（μL）空白孔测定孔样本020去离子水200Assay Buffer1401406-Phosphogluconic Acid Working Regent2020NADP⁺ Working Regent20203.4 混匀后，于340nm处测定3min内吸光值变化，空白孔第10s吸光值记为A1，第190s吸光值记为A2，ΔA空白=A2-A1；测定孔第10s吸光值记为A3，第190s吸光值记为A4，ΔA测定=A4-A3。 注意：实验之前建议选择2-3个预期差异大的样本做预实验。如果ΔA测定小于0.002可适当加大样本量。如果ΔA测定大于0.6，样本可用Assay Buffer进一步稀释，计算结果乘以稀释倍数，或减少提取用样本量。4、结果计算 注意：我们为您提供的计算公式，包括推导过程计算公式和简洁计算公式。两者完全相等。建议以加粗的简洁计算公式为最终计算公式。 4.1 使用 96 孔 UV 板测定的计算公式 (1) 按照液体体积计算 每毫升液体样本每分钟催化产生1μmol NADPH的酶量为1U。 G6PDH 酶活性(U/mL)=[(ΔA测定-ΔA空白)×V反总÷(ε×d)×10⁶]÷V样÷T=1.0718×(ΔA测定-ΔA空白) (2) 按蛋白浓度计算 每毫克蛋白每分钟催化产生1μmol NADPH的酶量为1U。 G6PDH 酶活性(U/mg prot)=[(ΔA测定-ΔA空白)×V反总÷(ε×d)×10⁶]÷(Cpr×V样)÷T=1.0718×(ΔA测定-ΔA空白)÷Cpr (3) 按样本鲜重计算 每克组织每分钟催化产生1μmol NADPH 的酶量为1U。 G6PDH 酶活性(U/g)=[(ΔA测定-ΔA空白)×V反总÷(ε×d)×10⁶]÷(V样÷V样总×W)÷T=1.0718×(ΔA测定-ΔA空白)÷W (4) 按细菌或细胞密度计算 每1万个细菌或细胞每分钟生成1μmol NADPH定义为一个酶活力单位。 G6PDH 酶活性(μmol/min/10⁴)＝[(ΔA测定-ΔA空白)×V反总÷(ε×d)×10⁶]÷(2000×V样÷V样总)÷T=0.0005359×(ΔA测定-ΔA空白)参数说明： ΔA测定：A4-A3； ΔA空白：A2-A1； ε：NADPH 摩尔消光系数，6.22×10³ L/mol/cm； d：96孔UV板光径，0.5 cm； 10⁶：1 mol=10⁶ μmol； V样：加入样本体积，0.02mL； V反总：反应体系总体积，0.0002L； Cpr：样本蛋白质浓度，mg/mL； V样总：样本制备加入Assay Buffer体积，1mL； W：样本鲜重，g； T：反应时间，3min； 2,000：细菌或细胞总数，2,000万。4.2 使用微量石英比色皿测定的计算公式将上述计算公式中的光径d: 0.5cm调整为d: 1cm进行计算即可。注意事项1、正式检测前，建议选择2-3个预期差异较大的样本进行预实验。2、本产品仅供科学研究使用，不适用于临床诊断。为了您的安全和健康，请穿实验服并戴一次性手套操作。
  Glucose-6-Phosphate Dehydrogenase (G6PD or G6PDH) (EC 1.1.1.49) is a cytosolic enzyme that catalyzes a chemical reaction. This enzyme participates in the pentose phosphate pathway, a metabolic pathway that supplies reducing power to cells (such as red blood cells) by maintaining NADPH levels. NADPH, in turn, maintains glutathione levels in these cells, protecting red blood cells from oxidative damage caused by compounds like hydrogen peroxide.  Assay Principle:G6PDH present in the sample converts NADP⁺ to NADPH. NADPH has a characteristic absorption peak at 340 nm. The absorbance value of NADPH is proportional to the G6PDH activity present in the sample.  Applicable Samples: Serum (Plasma), Animal/Plant Tissues, Cells, BacteriaG1506775Component48T96TStorageG1506775AAssay Buffer70 mL70 mL×22-8℃G1506775B6-Phosphogluconic Acid1EA1EA2-8℃. Store in the dark.G1506775CNADP⁺1EA1EA2-8℃. Store in the dark.Note: It is recommended to perform a pilot experiment with 2-3 samples expected to have significant differences before formal testing.Required Materials Not Provided1. Microplate reader or UV spectrophotometer (capable of measuring absorbance at 340 nm)2. 96-well UV microplate or micro quartz cuvettes, Adjustable pipettes and tips3. Deionized water4. Homogenizer (for tissue samples)Experimental Procedure1. Reagent PreparationReagent NamePreparation StepsNotes & StorageAssay BufferReady-to-use; equilibrate to room temperature before use.Store at 4°C.6-Phosphogluconic Acid Working RegentBefore use, dissolve the vial contents in 2.4 mL Assay Buffer. Mix uniformly.Keep on ice during use. Store prepared solution at 4°C protected from light for up to 1 week.NADP⁺ Working RegentBefore use, dissolve the vial contents in 2.4 mL Assay Buffer. Mix uniformly.Keep on ice during use. Store prepared solution at 4°C protected from light for up to 1 week.2. Sample Preparation2.1 Animal TissueWeigh about 0.1 g of tissue.Add 1 mL of Assay Buffer and homogenize in an ice bath.Centrifuge at 8,000 g, 4°C for 10 minutes.Collect the supernatant and keep on ice for assay.2.2 Plant TissueWeigh about 0.1 g of tissue.Add 1 mL of Assay Buffer, mash, and homogenize in an ice bath.Centrifuge at 8,000 g, 4°C for 10 minutes.Collect the supernatant and keep on ice for assay.2.3 Cells or BacteriaCollect 20 million cells or bacteria into a centrifuge tube.Wash cells with cold PBS, centrifuge at 12,000 g, 4°C for 1 min, discard supernatant.Add 1 mL of Assay Buffer.Sonicate cells or bacteria on ice for 5 minutes (20% power or 200 W, 3s pulse, 7s interval, repeat 30 times).Centrifuge at 8,000 g, 4°C for 10 minutes.Collect the supernatant and keep on ice for assay.2.4 Serum (Plasma) and Other Liquid SamplesAssay directly.Note: If measuring protein concentration is required, the use of the Aladdin B665595 BCA Protein Quantification Kit or R1491648 Ready-to-Use BCA Protein Quantification Kit is recommended.3. Assay Procedure3.1 Instrument PreparationPreheat the microplate reader or UV spectrophotometer for at least 30 minutes. Set the wavelength to 340 nm.Zero the UV spectrophotometer with deionized water.3.2 Buffer Pre-warmingPre-warm the Assay Buffer in a 25°C or 37°C water bath for at least 30 minutes.3.3 Assay SetupUse a 96-well UV plate. Add reagents as follows:Reagent (μL)Blank WellTest WellSample020Deionized Water200Assay Buffer1401406-Phosphogluconic Acid Working Regent2020NADP⁺ Working Regent20203.4 MeasurementMix well after addition.Measure the absorbance change at 340 nm over 3 minutes.For the Blank well: Record absorbance at 10 seconds as A₁, and at 190 seconds as A₂. Calculate ΔAblank = A₂ - A₁. For the Test well: Record absorbance at 10 seconds as A₃, and at 190 seconds as A₄. Calculate ΔAtest = A₄ - A₃. Note: A pilot experiment with 2-3 variable samples is recommended. If ΔAtest is less than 0.002, consider increasing the sample amount. If ΔAtest is greater than 0.6, dilute the sample further with Assay Buffer and multiply the result by the dilution factor, or reduce the sample amount used for extraction.4. Calculation of ResultsNote: We provide both the derived formula and the simplified formula for your convenience. They are mathematically equivalent. The bolded simplified formula is recommended for final calculations.4.1 Calculation using a 96-well UV Plate(1) Based on Liquid Volume:Definition of Unit (U): One unit is the amount of enzyme that catalyzes the production of 1 μmol of NADPH per minute per milliliter of liquid sample.Derived Formula: G6PDH Activity (U/mL) = [(ΔAtest - ΔAblank) × Vtotal reaction ÷ (ε × d) × 10⁶] ÷ Vsample ÷ T Simplified Formula: G6PDH Activity (U/mL) = 1.0718 × (ΔAtest - ΔAblank)(2) Based on Protein Concentration:Definition of Unit (U): One unit is the amount of enzyme that catalyzes the production of 1 μmol of NADPH per minute per milligram of protein.Derived Formula: G6PDH Activity (U/mg prot) =[(ΔAtest - ΔAblank) × Vtotal reaction ÷ (ε × d) × 10⁶] ÷ (Cpr × Vsample) ÷ T Simplified Formula: G6PDH Activity (U/mg prot) = 1.0718 × (ΔAtest - ΔAblank) ÷ Cpr(3) Based on Sample Fresh Weight:Definition of Unit (U): One unit is the amount of enzyme that catalyzes the production of 1 μmol of NADPH per minute per gram of tissue (fresh weight).Derived Formula: G6PDH Activity (U/g) =[(ΔAtest - ΔAblank) × Vtotal reaction ÷ (ε × d) × 10⁶] ÷ (Vsample ÷ Vtotal extract × W) ÷ T Simplified Formula: G6PDH Activity (U/g) = 1.0718 × (ΔAtest - ΔAblank) ÷ W(4) Based on Bacterial or Cell Density:Definition of Unit: One unit of enzyme activity is defined as the amount that generates 1 μmol of NADPH per minute per 10⁴ bacteria or cells.Derived Formula: G6PDH Activity (μmol/min/10⁴) =[(ΔAtest - ΔAblank) × Vtotal reaction ÷ (ε × d) × 10⁶] ÷ (2000 × Vsample ÷ Vtotal extract) ÷ T Simplified Formula: G6PDH Activity (μmol/min/10⁴) = 0.0005359 × (ΔAtest - ΔAblank)Parameter Definitions: ΔAtest: A₄ - A₃ (The change in absorbance for the Test well between 190s and 10s) ΔAblank: A₂ - A₁ (The change in absorbance for the Blank well between 190s and 10s) ε: Molar extinction coefficient of NADPH, 6.22 × 10³ L/mol/cm d: Light path of the 96-well UV plate, 0.5 cm 10⁶: Conversion factor (1 mol = 10⁶ μmol) Vsample: Volume of sample added to the reaction well, 0.02 mL Vtotal reaction: Total volume of the reaction mixture, 0.0002 L Cpr: Sample protein concentration, mg/mL Vtotal extract: Total volume of Assay Buffer added during sample preparation, 1 mL W: Sample fresh weight, g T: Reaction time, 3 min 2,000: Total number of bacteria or cells, in units of 10⁴ (e.g., 20 million cells = 2000 × 10⁴)4.2 Calculation using a Micro Quartz CuvetteUse the formulas above but adjust the light path d from 0.5 cm to 1 cm.Precautions1. It is recommended to perform a pilot experiment with 2-3 samples expected to have significant differences before formal testing.2. This product is for research use only. Not for use in diagnostic procedures. For your safety and health, please wear lab coats and disposable gloves during operation.