BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
2-8°C储存,避光。低温运输 。请查阅批次 COA 获取详细规格。
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
乳酸脱氢酶 (Lactatedehydrogenase，LDH) 是一种糖酵解酶，广泛存在于动物、植物、微生物和培养细胞中，其中以肾脏含量较高。LDH是糖酵解途径的末端酶，催化丙酮酸与乳酸之间的可逆反应，伴随着NAD⁺/NADH之间的互变。根据其催化底物乳酸构型的不同，可分为D-乳酸脱氢酶 (D-LDH，EC1.1.1.28) 与L-乳酸脱氢酶 (L-LDH，EC1.1.1.27)。
检测原理：D-LDH催化NAD⁺氧化D-乳酸生成丙酮酸，丙酮酸进一步与2,4-二硝基苯肼作用生成丙酮酸二硝基苯腙，在碱性溶液中显棕红色，颜色深浅与丙酮酸浓度成正比。
产品组份
D1505517 | 组分 | 物理外观 | 48 T | 96 T | 储存条件 | 单次使用量 |
D1505517A | 提取液 | 液体 | 70 mL | 70 mL×2 | 2-8℃. | 按需 |
D1505517B | 试剂一 | 液体 | 7 mL | 14 mL | 2-8℃. | 50 μL |
D1505517C | 试剂二 | 固体 | 17.3 mg | 34.5 mg | 2-8℃. 避光保存. | 10 μL |
D1505517D | 试剂三 | 液体 | 7 mL | 14 mL | 2-8℃. | 50 μL |
D1505517E | 试剂四 | 液体 | 20 mL | 40 mL | 2-8℃. | 150 μL |
D1505517F | 标准品(100μmol/mL) | 液体 | 1 mL | 1 mL | 2-8℃. 避光保存. | 按需 |
自备仪器和试剂
酶标仪或可见分光光度计(能测450nm处的吸光度)、96孔板或微量玻璃比色皿、可调节式移液枪及枪头、恒温水浴锅、制冰机、离心机、去离子水、匀浆器（如果是组织样本）
实验流程
1. 试剂准备
试剂 | 试剂准备 | 注意事项 |
提取液 | 即用型，使用前平衡到室温 | 2-8℃ |
试剂一 | 即用型，使用前平衡到室温 | 2-8℃ |
试剂二工作液 | 临用前配制；48T加入1.3mL去离子水，96T加入2.6mL去离子水，充分溶解待用。 | 用不完的试剂分装后-20℃避光保存1个月，禁止反复冻融。 |
试剂三 | 即用型，使用前平衡到室温 | 2-8℃ |
试剂四 | 即用型，使用前平衡到室温 | 2-8℃ |
标准品 | 即用型，使用前平衡到室温 | 2-8℃ |
2. 标准品制备
2.1. 使用100μmol/mL标准品，按照下表所示，进一步稀释标准品：
序号 | 标准品体积 | 提取液体积（µL） | 浓度（μmol/mL） |
Std.1 | 20µL of 100μmol/mL | 980 | 2 |
Std.2 | 100µL of Std.1 | 100 | 1 |
Std.3 | 100µL of Std.2 | 100 | 0.5 |
Std.4 | 100µL of Std.3 | 100 | 0.25 |
Std.5 | 100µL of Std.4 | 100 | 0.125 |
Std.6 | 100µL of Std.5 | 100 | 0.063 |
Std.7 | 100µL of Std.6 | 100 | 0.031 |
Blank | 0 | 100 | 0 |
注意：每次实验都要做一次标准品检测，制作标曲；稀释后的标准品溶液不稳定，必须在4小时内使用。
3. 样本制备
3.1. 细菌或细胞：先收集细菌或细胞到离心管内，离心后弃上清；按照细菌或细胞数量(10⁴个)：提取液体积(mL)为500-1000: 1的比例(建议500万细菌或细胞加入1mL 提取液)加入提取液，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)，然后8000g，4℃离心10min，取上清液，置冰上待测。
3.2. 组织：按照组织质量(g)：提取液体积(mL)为1: 5-10的比例(建议称取约0.1g组织，加入1mL 提取液)加入提取液，进行冰浴匀浆。8000 g，4℃离心10min，取上清，置冰上待测。
3.3. 血清(浆)：直接检测。
4. 实验步骤
4.1. 酶标仪或可见分光光度计预热30min，调节波长到450nm。可见分光光度计用去离子水调零。
4.2. 操作表(下述操作在1.5mL EP管中进行)：
试剂 | 测定管 (μL) | 对照管 (μL) | 标准管 (μL) |
待测样本 | 10 | 10 | - |
标准品 | - | - | 10 |
试剂一 | 50 | 50 | 50 |
试剂二工作液 | 50 | 50 | 50 |
去离子水 | 0 | 10 | 10 |
充分混匀，37℃水浴15min。 | |||
试剂三 | 50 | 50 | 50 |
充分混匀，37℃水浴15min。 | |||
试剂四 | 150 | 150 | 150 |
4.3. 充分混匀，室温静置30min，取200μL转移至微量玻璃比色皿或96孔板中于450nm处测定吸光度，分别记为A测定、A对照 、 A标准 、 A空白，计算ΔA测定 = A测定 - A对照 、ΔA标准 = A标准 - A空白 。
注意：空白管和标准曲线只需测定1次，每个测定管需设置一个对照管。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0.01，可适当增大样本量；如果ΔA测定 大于0.5，样本可用提取液进一步稀释，计算结果乘以稀释倍数，或减少提取用样本量。
5. 结果计算
5.1. 标准曲线的绘制：以标准溶液浓度为x轴，ΔA标准为y轴，绘制标准曲线，得到标准方程，将ΔA测定 带入方程得到x(μmol/mL)。
5.2. D-LDH活性计算
5.2.1. 按样本体积计算：单位的定义：每mL血清(浆)每分钟催化产生1nmol丙酮酸定义为一个酶活力单位。 D-LDH活性(U/mL)=x×V样÷V样÷T×10³=66.67×x
5.2.2. 按样本质量计算：单位的定义：每g组织每分钟催化产生1nmol丙酮酸定义为一个酶活力单位。D-LDH活性(U/g质量)=x×V样÷(W÷V样总×V样 )÷T×10³=66.67×x÷W
5.2.3. 按细菌或细胞数量计算：单位的定义：每10⁴个细菌或细胞每分钟催化产生1nmol丙酮酸定义为一个酶活力单位。D-LDH活性 (U/10⁴)=x×V样÷(N÷V样总×V样 )÷T×10³=66.67×x÷N
参数说明
V样：反应体系中加入的样本体积，0.01mL；
V样总：加入的提取液体积，1mL；
T：反应时间，15min；
W：样本质量，g；
N：细胞或细菌数量，以万计；
10³：单位换算系数，1μmol/mL=10³ nmol/mL。
结果展示
标准曲线
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。
Lactate dehydrogenase (LDH) is a glycolytic enzyme widely found in animals, plants, microorganisms, and cultured cells, with relatively high content in the kidneys. LDH is the terminal enzyme of the glycolytic pathway, catalyzing the reversible reaction between pyruvate and lactate, accompanied by the interconversion of NAD⁺/NADH. Based on the stereospecificity for the lactate substrate, LDH can be classified into D-lactate dehydrogenase (D-LDH, EC 1.1.1.28) and L-lactate dehydrogenase (L-LDH, EC 1.1.1.27).
Detection Principle: D-LDH catalyzes the oxidation of D-lactate by NAD⁺ to generate pyruvate. Pyruvate further reacts with 2,4-dinitrophenylhydrazine to form pyruvate dinitrophenylhydrazone, which exhibits a brown-red color in an alkaline solution. The color intensity is proportional to the pyruvate concentration.
Kit Contents
D1505517 | Components | Appearance | 48 T | 96 T | Storage | Quantity Per Test |
D1505517A | Extraction Buffer | Liquid | 70 mL | 70 mL×2 | 2-8℃. | As needed |
D1505517B | Reagent Ⅰ | Liquid | 7 mL | 14 mL | 2-8℃. | 50 μL |
D1505517C | Reagent Ⅱ | Powder | 17.3mg | 34.5mg | 2-8℃. Store in the dark. | 10 μL |
D1505517D | Reagent Ⅲ | Liquid | 7 mL | 14 mL | 2-8℃. | 50 μL |
D1505517E | Reagent Ⅳ | Liquid | 20 mL | 40 mL | 2-8℃. | 150 μL |
D1505517F | Standard (100μmol/mL) | Liquid | 1 mL | 1 mL | 2-8℃. Store in the dark. | As needed |
User-Prepared Instruments and Reagents:
1. Microplate reader or visible spectrophotometer (capable of measuring absorbance at 450 nm)
2. 96-well plate or micro glass cuvettes, adjustable micropipettes and tips
3. Constant temperature water bath, ice maker, centrifuge
4. Deionized water
5. Homogenizer (for tissue samples)
Experimental Procedure
1. Reagent Preparation
Reagent | Preparation | Blank Well |
Extraction Buffer | Ready-to-use; equilibrate to room temperature before use. | Store at 2-8°C |
Reagent Ⅰ | Ready-to-use; equilibrate to room temperature before use. | Store at 2-8°C |
Working Reagent Ⅱ | Prepare before use. For 48T, add 1.3 mL deionized water; for 96T, add 2.6 mL deionized water. Dissolve completely | Unused reagent can be aliquoted and stored at -20°C protected from light for one month. Avoid repeated freeze-thaw cycles. |
Reagent Ⅲ | Ready-to-use; equilibrate to room temperature before use. | Store at 2-8°C |
Reagent Ⅳ | Ready-to-use; equilibrate to room temperature before use. | Store at 2-8°C |
Standard | Ready-to-use; equilibrate to room temperature before use. | Store at 2-8°C protected from light |
2. Standard Preparation
2.1. Using the 100 μmol/mL standard stock, prepare a dilution series as shown in the table below:
Tube | Standard Volume | Extraction Buffer Volume (μL) | Concentration (μmol/mL) |
Std.1 | 20µL of 100μmol/mL | 980 | 2 |
Std.2 | 100µL of Std.1 | 100 | 1 |
Std.3 | 100µL of Std.2 | 100 | 0.5 |
Std.4 | 100µL of Std.3 | 100 | 0.25 |
Std.5 | 100µL of Std.4 | 100 | 0.125 |
Std.6 | 100µL of Std.5 | 100 | 0.063 |
Std.7 | 100µL of Std.6 | 100 | 0.031 |
Blank | 0 | 100 | 0 |
Note: The standard curve must be generated with each experiment. Diluted standard solutions are unstable and must be used within 4 hours.
3. Sample Preparation
3.1. Bacteria or Cells: Collect bacteria or cells into a centrifuge tube, centrifuge, discard supernatant. Add Extraction Buffer at a ratio of bacteria/cell count (10⁴) to volume (mL) between 500:1 and 1000:1 (recommended: 5 million bacteria/cells in 1 mL Extraction Buffer). Sonicate in an ice bath for 5 min (power 20% or 200W, pulse 3s on, 7s off, repeat 30 times). Centrifuge at 8000 g, 4°C for 10 min. Collect supernatant and keep on ice for assay.
3.2. Tissue: Add Extraction Buffer at a tissue mass (g) to volume (mL) ratio between 1:5 and 1:10 (recommended: weigh approx. 0.1 g tissue, add 1 mL Extraction Buffer). Homogenize in an ice bath. Centrifuge at 8000 g, 4°C for 10 min. Collect supernatant and keep on ice for assay.
3.3. Serum (Plasma): Assay directly.
4. Assay Steps
4.1. Preheat the microplate reader or visible spectrophotometer for 30 min. Set wavelength to 450 nm. For spectrophotometers, zero with deionized water.
4.2. Assay Procedure (perform in 1.5 mL microcentrifuge tubes):
Reagent | Test Tube (μL) | Control Tube (μL) | Standard Tube (μL) |
Test Sample | 10 | 10 | - |
Standard | - | - | 10 |
Reagent Ⅰ | 50 | 50 | 50 |
Working Reagent Ⅱ | 10 | - | - |
Deionized Water | 0 | 10 | 10 |
Mix thoroughly, incubate at 37°C for 15 min. | |||
Reagent Ⅲ | 50 | 50 | 50 |
Mix thoroughly, incubate at 37°C for 15 min. | |||
Reagent Ⅳ | 150 | 150 | 150 |
4.3. Mix thoroughly, let stand at room temperature for 30 min. Transfer 200 μL to a micro glass cuvette or 96-well plate. Measure absorbance at 450 nm, recorded as Atest、Acontrol 、 Astandard 、 Ablank， Calculate ΔAtest = Atest - Acontrol 、ΔAstandard = Astandard - Ablank.
Note: The Blank and Standard Curve tubes need only be set up once. Each test sample requires a control tube. A preliminary test with 2-3 samples showing expected significant differences is recommended. If ΔAtest < 0.01, increase sample volume appropriately. If ΔAtest > 0.5, dilute sample further with Extraction Buffer (multiply result by dilution factor) or reduce sample amount used for extraction.
5. Calculation of Results
5.1. Standard Curve Plotting: Plot the standard concentration (x-axis) against ΔAstandard (y-axis) to generate the standard curve and obtain the standard equation. Substitute ΔAtest into the equation to obtain x (μmol/mL).
5.2. D-LDH Activity Calculation
5.2.1. Based on Sample Volume: Unit Definition: One unit of enzyme activity is defined as the amount that produces 1 nmol of pyruvate per minute per mL of serum (plasma). Derived Formula: D-LDH Activity (U/mL) = x × Vsample ÷ Vsample ÷ T × 10³. Simplified Formula: D-LDH Activity (U/mL) = 66.67 × x
5.2.2. Based on Sample Mass: Unit Definition: One unit of enzyme activity is defined as the amount that produces 1 nmol of pyruvate per minute per gram of tissue. Derived Formula: D-LDH Activity (U/g mass) = x × Vsample ÷ (W ÷ Vtotal sample × Vsample) ÷ T × 10³ . Simplified Formula: D-LDH Activity (U/g mass) = 66.67 × x ÷ W
5.2.3. Based on Bacterial or Cell Count: Unit Definition: One unit of enzyme activity is defined as the amount that produces 1 nmol of pyruvate per minute per 10⁴ bacteria or cells. Derived Formula: D-LDH Activity (U/10⁴) = x × Vsample ÷ (N ÷ Vtotal sample × Vsample) ÷ T × 10³. Simplified Formula: D-LDH Activity (U/10⁴) = 66.67 × x ÷ N
Parameter Definitions:
Vsample: Volume of sample added to the reaction system (0.01 mL)
Vtotal sample: Volume of Extraction Buffer added (1 mL)
T: Reaction time (15 min)
W: Sample mass (g)
N: Number of cells or bacteria (in units of 10⁴)
10³: Unit conversion factor (1 μmol/mL = 10³ nmol/mL)
Result Presentation
Standard curve
Notes
1. Before formal testing, it is recommended to perform a preliminary test with 2-3 samples expected to have significant differences.
2. This product is for research use only. Not for use in clinical diagnosis. For your safety and health, please wear a lab coat and disposable gloves during operation.
D1505517 | 组分名称 | 物理外观 | 48 T | 96 T | 储存 | 单次测试使用量 |
D1505517A | 提取液 | 液体 | 70 mL | 70 mL×2 | 2-8℃. | 按需 |
D1505517B | 试剂一 | 液体 | 7 mL | 14 mL | 2-8℃. | 50 μL |
D1505517C | 试剂二 | 粉末 | 17.3mg | 34.5mg | 2-8℃. 避光 | 10 μL |
D1505517D | 试剂三 | 液体 | 7 mL | 14 mL | 2-8℃. | 50 μL |
D1505517E | 试剂四 | 液体 | 20 mL | 40 mL | 2-8℃. | 150 μL |
D1505517F | 标准品(100μmol/mL) | 液体 | 1 mL | 1 mL | 2-8℃. 避光 | 按需 |
注：试剂二溶解后，剩余部分需分装并置于-20℃避光保存。
危险品化学品经营许可证（带存储）