碱性磷酸酶检测试剂盒
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产品编号 ： P0321M
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¥ 1209.00 元
产品包装:
100次 500次
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碧云天生产的碱性磷酸酶检测试剂盒(Alkaline Phosphatase Assay Kit)是一种用于快速、便捷地检测细胞或组织样品的裂解或匀浆产物的上清液、血清、血浆、尿液等样品中内源性的碱性磷酸酶活性的试剂盒。
碱性磷酸酶(Alkaline Phosphatase, AP/ALP/AKP/ALKP/ALPase/Alk Phos)也称碱性磷酸酯酶(EC 3.1.3.1)，可以在碱性条件下催化磷酸酯键的水解。哺乳动物中，肝脏、胆管、肾脏、骨头和胎盘中的碱性磷酸酶活性比较高。常见的碱性磷酸酶包括肠道碱性磷酸酶(alkaline phosphatase, intestinal, ALPI)、非组织特异性碱性磷酸酶(alkaline phosphatase, tissue-nonspecific isozyme, ALPL)和胎盘碱性磷酸酶(alkaline phosphatase, placental type, 也称placental alkaline phosphatase, PLAP)。常见的小牛肠碱性磷酸酶(Calf Intestinal Alkaline Phosphatase, CIAP/CIP)被广泛用于二抗等的标记最终用于蛋白和核酸等的检测，也常用于DNA或RNA 5'和3'末端的去磷酸化(去单磷酸化)，特别是质粒的5'末端去磷酸化以避免质粒自连等。
干细胞，如iPS中，碱性磷酸酶的活性很高，常被用作iPS成功诱导的标志。另外，分化的结肠癌细胞中碱性磷酸酶的活性也会显著升高，被当作结肠癌细胞分化程度定性和定量的指标。此外，血清中碱性磷酸酯酶的升高，被称作高碱性磷酸酶血症(hyperalkalinephosphatasemia)，被认为和恶性胆管阻塞(malignant biliary obstruction)、原发性胆管硬化(primary biliary cirrhosis)、原发性硬化胆管炎(primary sclerosing cholangitis)、肝淋巴瘤(hepatic lymphoma)和肝肉瘤(hepatic sarcoidosis)等肝胆疾病密切相关。血清中碱性磷酸酶活性升高还和骨头生成密切相关，因为碱性磷酸酶是成骨细胞的副产物。血清中碱性磷酸酶活性过低也和一些疾病相关。儿童和孕妇血清中的碱性磷酸酶活性较普通人高一些。血清中碱性磷酸酶活性范围在20-140U/L。
除胎盘碱性磷酸酶(alkaline phosphatase placental isoform)以外，其它的内源性碱性磷酸酶加热后易失活。
本试剂盒可以检测细胞或组织样品的裂解或匀浆产物的上清液、血浆、血清、尿液或纯化的酶样品等中的碱性磷酸酶活性。
Para-nitrophenyl phosphate (pNPP)是一种常用的磷酸酶显色底物，在碱性条件下，可在碱性磷酸酶作用下生成para-nitrophenol。para-nitrophenol(p-nitrophenol)在碱性条件下，呈黄色产物，可以在400-415nm检测吸光度。产物黄色越深，说明碱性磷酸酶检活性越高，反之则酶活性越低。据此通过比色分析就可以计算出碱性磷酸酶活性水平。
包括标准品和空白对照，本试剂盒P0321S包装共可进行100个样品的检测，P0321M包装共可进行500个样品的检测。
包装清单：

产品编号 产品名称 包装
P0321S-1 检测缓冲液 15ml
P0321S-2 显色底物 2管
P0321S-3 p-nitrophenol溶液(10mM) 0.1ml
P0321S-4 反应终止液 12ml
— 说明书 1份

产品编号 产品名称 包装
P0321M-1 检测缓冲液 65ml
P0321M-2 显色底物 5管
P0321M-3 p-nitrophenol溶液(10mM) 0.5ml
P0321M-4 反应终止液 60ml
— 说明书 1份

保存条件：
-20℃保存，一年有效。其中显色底物和p-nitrophenol溶液需避光保存。
注意事项：
如果希望进行酶活性的绝对定量，进行酶反应时必须注意精确计时。此时推荐采用孵育30分钟等较长的时间，以减小操作过程中的时间误差。同时如果样品中酶活性较高，则可以预先适当稀释样品。
样品溶液中须避免出现EDTA、氟离子、柠檬酸盐等碱性磷酸酶的抑制剂。
检测缓冲液和p-nitrophenol溶液对人体有害，操作时请小心，并注意有效防护以避免直接接触人体或吸入体内。反应终止液有腐蚀性，操作时请小心，并注意有效防护以避免直接接触人体或腐蚀其他物品。
本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。
为了您的安全和健康，请穿实验服并戴一次性手套操作。

使用说明：
1. 试剂准备：将所有试剂取出，恢复至室温使用。
a. 显色底物溶液：对于P0321S包装，请取一管显色底物，溶解于2.5ml的检测缓冲液中(可先用1ml检测缓冲液进行溶解，充分溶解和混匀后，转移至15ml离心管，再加入1.5ml检测缓冲液)，充分溶解和混匀，冰上放置。对于P0321M包装，请取一管显色底物，溶解于5ml的检测缓冲液中(可先用2ml检测缓冲液进行溶解，充分溶解和混匀后，转移至15ml离心管，再加入3ml检测缓冲液)，充分溶解和混匀，冰上放置。新鲜配制的显色底物溶液需在6小时内使用。
b. 标准品工作液：取10μl p-nitrophenol溶液(10mM)，用检测缓冲液稀释至0.2ml，最终浓度为0.5mM。
2. 样品准备：
a. 细胞或组织裂解液的准备：采用适当细胞或组织裂解液裂解细胞或组织，建议使用碧云天的P0013J Western及IP细胞裂解液(无抑制剂)裂解相关样品。如果有必要需进行适当匀浆，随后离心取上清，用于碱性磷酸酶的检测。注意：裂解液中不能含有磷酸酶抑制剂。样品可以-80℃冻存，但需避免反复冻融。
b. 血浆、血清和尿液的准备：血浆和血清按照常规方法制备后可以直接用于本试剂盒的测定，但为了消除样品本身颜色的干扰，需设置加了血浆或血清但不加底物的对照。血浆制备时不能用含EDTA和柠檬酸盐的抗凝管。尿液通常也可以直接用于测定。上述样品可以-80℃冻存，但需避免反复冻融。
c. 样品的稀释：如果样品中含有较高活性的碱性磷酸酶，可以使用原有的裂解液或PBS等进行稀释，也可以采用试剂盒中的检测
缓冲液进行稀释。如果使用试剂盒中提供的检测缓冲液进行稀释，需注意保留足够的检测缓冲液用于试剂盒的检测过程。
3. 参考下表使用96孔板设置空白对照孔、标准品孔和样品孔。标准品的用量分别为4、8、16、24、32和40微升，样品通常可以直接加50微升。如果样品中的碱性磷酸酶活性过高，可以减少样品用量或适当稀释后再进行测定。

空白对照(Blank) 标准品(Standard) 样品(Sample)
检测缓冲液 50μl (100-x)μl (50-y)μl
显色底物 50μl － 50μl
样品 － － yμl
标准品工作液 － xμl －

4. 用枪头轻轻吹打混匀，也可借助摇床进行混匀。
5. 37℃孵育5-10分钟。(说明：待测样品中碱性磷酸酶活性较低时，可适当延长孵育时间至30分钟)
6. 每孔加入100μl反应终止液终止反应。此时，标准品或有碱性磷酸酶活性的孔会呈现不同深浅的黄色。
7. 在405nm测定吸光度。如果不能测定405nm，也可以在400-415nm范围内检测吸光度。如果不能立即测定，可以在数小时内完成测定，所显现的黄色在数小时内稳定。
8. 碱性磷酸酶活性单位的定义：在pH9.8的diethanolamine(DEA)缓冲液中，37℃条件下，每分钟水解para-nitrophenyl phosphate显色底物产生1微摩尔p-nitrophenol所需的碱性磷酸酶的量定义为一个酶活力单位，也被称作一个DEA酶活力单位。在pH9.6的甘氨酸缓冲液中，25℃条件下，每分钟水解para-nitrophenyl phosphate显色底物产生1微摩尔p-nitrophenol所需的碱性磷酸酶的量定义为一个酶活力单位，也被称作一个Glycine酶活力单位。一个Glycine酶活力单位约相当于3个DEA酶活力单位。本试剂盒测定的是DEA酶活力单位。
9. 根据酶活性定义，计算出样品中的碱性磷酸酶活性。
相关产品：

产品编号 产品名称 包装
P0013J Western及IP细胞裂解液(无抑制剂) 100ml
P0321S 碱性磷酸酶检测试剂盒 100次
P0321M 碱性磷酸酶检测试剂盒 500次
P0326 酸性磷酸酶检测试剂盒 120次
P0329 胎盘碱性磷酸酶检测试剂盒 100次
P0332 抗酒石酸酸性磷酸酶检测试剂盒 120次
P0335 抗氟离子酸性磷酸酶检测试剂盒 120次


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P0321S
100次
碱性磷酸酶检测试剂盒
C3206
共100ml
BCIP/NBT碱性磷酸酯酶显色试剂盒
P0322S
100次
碱性磷酸酶检测试剂盒(荧光法)
C0148S
50次
成骨细胞矿化结节染色试剂盒(茜素红S法)
C3250S
共100ml
多能干细胞碱性磷酸酶显色试剂盒
P0322M
500次
碱性磷酸酶检测试剂盒(荧光法)
P0013J
100ml
Western及IP细胞裂解液(无抑制剂)
P0326
120次
酸性磷酸酶检测试剂盒
D7027
200U
BeyoAP Alkaline Phosphatase
P0329
100次
胎盘碱性磷酸酶检测试剂盒
P0332
120次
抗酒石酸酸性磷酸酶检测试剂盒
C0138-100ml
100ml
茜素红S染色液(2%, pH4.2)