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- 保存条件：
-20°C
- 保质期：
6个月
- 英文名：
Isocitrate Dehydrogenase Mitochondrial(ICDHm) Activity Assay Kit
- 库存：
999
- 供应商：
北京索莱宝科技有限公司
- CAS号：
BC2165-100T/48S
- 规格：
100管/48样
线粒体异柠檬酸脱氢酶（isocitrate dehydrogenase，ICDHm），广泛存在于动物、植物、微生物和培养细胞的线粒体中，与线粒体基因表达及线粒体其他的功能有关。异柠檬酸脱氢酶在生物体内有两种存在形式，以NAD为辅酶的NAD-依赖型异柠檬酸脱氢酶，和以NADP为辅酶的NADP-依赖型异柠檬酸脱氢酶。
异柠檬酸脱氢酶的主要功能，是在体内三羧酸循环中，催化异柠檬酸生成α-酮戊二酸，将NAD还原成NADH，通过测定α-酮戊二酸的生成量，可以计算出线粒体异柠檬酸脱氢酶活力高低。
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。如果样本吸光值不在测量范围内建议稀释或者增加样本量进行检测。
需自备的仪器和用品：
低温离心机、可见分光光度计/酶标仪、水浴锅/恒温培养箱、微量玻璃比色皿/96孔板、可调式移液枪、研钵/匀浆器、冰和蒸馏水。
操作步骤：
一、样本处理（可适当调整待测样本量，具体比例可以参考文献）
1. 称取约0.2 g组织或收集1000万细胞，加入1mL提取液一和10μL提取液二，用冰浴匀浆器或研钵匀浆。
2. 4℃，1000g离心10min。将上清液移至另一离心管中，4℃，11000g离心15min。
3. 上清液即胞浆提取物，可用于测定从线粒体泄漏的异柠檬酸脱氢酶（此步可选做，可以判断线粒体提取效果）。
4. 在沉淀中加入400μL提取液三和4μL提取液二，超声波破碎（功率300w，超声5秒，间隔9秒，4min），4℃，10000g离心10min，取上清液用于线粒体异柠檬酸脱氢酶活性测定，并且用于蛋白含量测定。
三、ICDHm活性计算
1、标准曲线绘制
以各个标准溶液的浓度为x轴，其对应的ΔA标准为y轴，绘制标准曲线，得到标准方程y=kx+b，将ΔA带入方程得到x(μmol/mL)。
2、酶活力计算
酶活定义：每mg组织蛋白在反应体系中每分钟产生1nmol α-酮戊二酸定义为一个酶活性单位。
ICDHm酶活力（U/mg prot）=x×V上清÷（Cpr×V上清）÷T×103=x÷Cpr×16.67
V上清：加入上清液体积，0.04mL；Cpr：样本蛋白浓度，mg/mL，需自行测定；V反总：反应体系总体积，0.2mL；T：反应时间，1h=60min；103：单位换算系数，1μmol =103nmol。
注意事项：
1、 为保证实验结果的准确性，需先取1-2个样做预实验，如果测定的吸光值过高（高于1），可用提取液三稀释上清液后再测定。计算结果时注意乘以稀释倍数。
2、 推荐使用样本蛋白浓度计算酶活，若用样本质量计算，则需加测胞浆提取物酶活，上清和沉淀酶活之和方为总酶活。
3、 测定蛋白浓度时，由于试剂一本身含有蛋白（约1mg/mL），所以测定时需扣除此部分蛋白。
4、 附：使用样本重量计算公式
A、上清（胞浆）中ICDHm活力计算：
酶活定义：每g组织在反应体系中每分钟产生1nmol α-酮戊二酸定义为一个酶活性单位。
ICDHm活性（U/g 质量）=x×V样÷(W×V样÷V提取)÷T×103=16.83×x÷W
V提取：加入提取液体积，1.01mL；V样：加入上清液体积，0.04mL；W：样本质量，g；T：反应时间，1h=60min；103：单位换算系数，1μmol =103nmol。
B、沉淀（线粒体）中ICDHm活力计算：
酶活定义：每g组织在反应体系中每分钟产生1nmol α-酮戊二酸定义为一个酶活性单位。
ICDHm活性（U/g 质量）=x×V样÷(W×V样÷V提取)÷T×103=6.73×x÷W
V提取：沉淀重悬时加入提取液体积，0.404mL；V样：加入上清液体积，0.04mL；W：样本质量，g；T：反应时间，1h=60min；103：单位换算系数，1μmol =103nmol。
C、样本ICDHm总活力计算：
样本ICDHm总活力即为上清（胞浆）中ICDHm活力与沉淀（线粒体）中ICDHm活力之和。
按样本质量计算：ICDHm（U/g 质量）=16.83×x÷W+6.73×x÷W
实验实例：
1、 取0.2g小鼠肾脏加入1.5 mL提取液一和15 μL提取液二，用冰浴匀浆器匀浆。4℃，1000g离心10min。将上清液移至另一离心管中，4℃，11000g离心15min。上清液即胞浆提取物，在沉淀中加入600μL提取液三和6μL提取液二，超声波破碎，4℃，10000g离心10min，取上清液，分别按操作步骤检测，用96孔板测得：胞浆ΔA测定=A测定管-A对照管=0.25-0.25=0，线粒体ΔA测定=A测定管-A对照管=0.433-0.279=0.154，带入标准曲线y=0.5533x+0.0319计算x值，按样本质量计算酶活得：
胞浆中ICDHm活性（U/g质量）=16.83×x÷W=0 U/g质量
线粒体中ICDHm活性（U/g质量）=6.73×x÷W=7.43 U/g质量
样本总ICDHm（U/g 质量）=16.83×x÷W+6.73×x÷W=7.43 U/g质量。
2、 取0.3g黑麦草加入1.5mL提取液一和15μL提取液二，用冰浴匀浆器匀浆。4℃，1000g离心10min。将上清液移至另一离心管中，4℃，11000g离心15min。上清液即胞浆提取物，上清液稀释2倍，在沉淀中加入600μL提取液三和6μL提取液二，超声波破碎，4℃，10000g离心10min，取上清液稀释2倍，分别按操作步骤检测，用96孔板测得：胞浆ΔA测定=A测定管-A对照管=0.377-0.34=0.037，线粒体ΔA测定=A测定管-A对照管=0.711-0.649=0.062，带入标准曲线y=0.5533x+0.0319计算x值，按样本质量计算酶活得：
胞浆中ICDHm活性（U/g质量）=16.83×x÷W×2=1.55 U/g质量
线粒体中ICDHm活性（U/g质量）=6.73×x÷W×2=3.66U/g质量
样本总ICDHm（U/g 质量）=16.83×x÷W×2+9.16×x÷W×2=5.21 U/g质量。
相关发表文献：
[1] Xiao Li,Qi Zhao,Jianni Qi,et al. lncRNA Ftx promotes aerobic glycolysis and tumor progression through the
PPARγ pathway in hepatocellular carcinoma. International Journal of Oncology. May 2018;(IF3.571)
参考文献：
[1] Igamberdiev A U, Gardeström P. Regulation of NAD-and NADP-dependent isocitrate dehydrogenases by reduction levels of pyridine nucleotides in mitochondria and cytosol of pea leaves[J]. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 2003, 1606(1-3): 117-125.
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类：· Serine proteases· Cysteine proteases· Aspartic acid proteases· Zinc metalloproteases图示：比色蛋白酶试验响应曲线。采用 Thermo Scientific Pierce 比色法蛋白酶检测试剂盒，通过与提供的胰蛋白酶标准品进行比较，测定 V-8 蛋白酶和颌下腺蛋白酶消化酪蛋白底物的活性。以上即为PTMs的概述（资料来自Thermo Fisher官网），欢迎留言评论
探针很容易被装载到细胞内。细胞内的活性氧可以氧化无荧光的DCFH生成有荧光的DCF。检测DCF的荧光就可以知道细胞内活性氧的水平。活性氧检测试剂盒[9]。荧光法检测线粒体产生和释放的反应性氧化物（ROS）[10]。羟基自由基的检测（二羟苄胺DHBAs和水杨酸盐Salicylate）[11] ROS was evaluated by the staining of 2’，7’- dichlorofluorescein diacetate（二氯荧光乙酰乙酸盐）.ROS production
化物质检测等。 美国著名细胞产品公司Cell BioLabs为您提供完备的细胞氧化应激分析解决方案，其产品包含蛋白质羰基化、硝基化损伤及晚期氧化蛋白产物（AOPP）分析，脂质过氧化的标志物壬烯(HNE)和丙二醛(MDA)以及8-异前列腺素F2a快速检测试剂盒，核酸DNA/RNA的常规损伤分析如8-OHdG/8-OHG和AP位点检测，还包括细胞水平的彗星分析和DNA双链断裂分析试剂盒；除此之外，还有多种抗氧化物的活性检测方案供您选择，如过氧化物歧化酶（SOD）、过氧化氢酶 （CAT）及ORAC指标
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