脂质氧化(MDA)检测试剂盒
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产品编号 ： S0131M
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¥ 1450.00 元
产品包装:
100次 500次
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碧云天的脂质氧化(MDA)检测试剂盒(Lipid Peroxidation MDA Assay Kit)采用一种基于MDA和硫代巴比妥酸(thiobarbituric acid,TBA)反应产生红色产物的显色反应，随后通过比色法用于对血浆、血清、尿液、动植物组织或细胞裂解液中MDA进行定量检测，广泛用于脂质氧化(lipid peroxidation)水平检测的试剂盒。
丙二醛(Malondialdehyde,MDA)是一种生物体脂质氧化的天然产物。动物或植物细胞发生氧化应激(oxidative stress)时，会发生脂质氧化。一些脂肪酸氧化后逐渐分解为一系列复杂的化合物，其中包括MDA。此时通过检测MDA的水平即可检测脂质氧化的水平，因此MDA的测定被广泛用作脂质氧化的指标。生物体内的一些其它生化反应也会产生MDA，例如thromboxane synthase也可以催化产生，但只要在测定时设置适当对照即可观察到脂质氧化水平的变化。
丙二醛在较高温度及酸性环境中可与TBA发生反应，形成红色的MDA-TBA加合物，相应的反应原理图如下：


MDATBAMDA-TBA Adduct

MDA-TBA加合物在535nm处有最大吸收，据此可以通过比色法进行检测。另外，MDA-TBA加合物也可以在535nm被激发产生最大发射波长553nm，据此也可以进行荧光检测。
特点：本试剂盒中采用了特殊的抗氧化剂，可以有效地抑制样品在检测过程中产生新的MDA，使检测更加准确。同时本检测试剂盒在检测过程中可以把部分MDA天然形成的聚丙二醛分解成MDA，使对脂质氧化的测定更加准确。
本试剂盒可以检测低达1μM的MDA，也可检测高达200μM的MDA (参考图1)。血浆、血清样品中的MDA含量通常在约2-4μM，尿液中的MDA含量通常在约5-30μM，在本试剂盒的检测范围内，可以直接用本试剂盒检测血浆、血清、尿液样品等。

图1. 不同浓度标准品使用本试剂盒的检测效果图。实测数据会因检测仪器等的不同而存在差异，图中数据仅供参考。
本试剂盒S0131S包装共可进行100次检测，S0131M包装共可进行500次检测。
包装清单：

产品编号 产品名称 包装　
S0131S-1 TBA 25mg
S0131S-2 TBA配制液 6.76ml
S0131S-3 TBA稀释液 15ml
S0131S-4 抗氧化剂 300μl
S0131S-5 标准品(1mM) 200μl
— 说明书 1份

产品编号 产品名称 包装　
S0131M-1 TBA 125mg
S0131M-2 TBA配制液 35ml
S0131M-3 TBA稀释液 75ml
S0131M-4 抗氧化剂 1.5ml
S0131M-5 标准品(1mM) 1ml
— 说明书 1份

保存条件：
-20℃保存，一年有效。S0131-1 TBA和S0131-4抗氧化剂需避光保存。S0131-1 TBA、S0131-2 TBA配制液和S0131-3 TBA稀释液可室温或4℃存放三个月。
注意事项：
醛、较高浓度的可溶性糖(例如250mM蔗糖)对反应有干扰，可溶性糖与TBA显色反应的产物在532nm也有吸收(最大吸收在450nm)。如果可溶性糖对测定有干扰，可以通过测定450nm作为参考波长进行双波长测定，消除其干扰。
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. 样品的准备：
a. 血浆、血清或尿液样品制备后可以直接用于MDA测定。
b. 组织或细胞可以使用PBS或碧云天的Western及IP细胞裂解液(P0013)等裂解液进行匀浆或裂解。对于组织，组织重量占匀浆液或裂解液的比例为10%；对于细胞，每100万细胞使用0.1ml裂解液或匀浆液。匀浆或裂解后，10,000g-12,000g离心10分钟取上清用于后续测定。对于一些特殊样品，离心不能获得澄清的上清溶液的，可以使用0.2微米孔径的过滤器过滤以获得澄清的样品溶液。匀浆或裂解等样品制备步骤宜在冰浴或4℃进行操作。
c. 对于组织或细胞样品，样品准备完毕后可以用BCA蛋白浓度测定试剂盒(P0009/P0010/P0010S/P0011/P0012/P0012S)测定蛋白浓度，以便于后续计算单位蛋白重量组织或细胞内的MDA含量。
d. 本试剂盒对于样品中的常见化学成分的兼容性参考下表：

试剂类别 化学成分 是否干扰
缓冲试剂 Borate (50mM) 否
HEPES (100mM) 否
Phosphate (100mM) 否
Tris (25mM) 否
去垢剂 CHAPS (≤1%) 否
Triton X-100 (≤1%) 否
Tween 20 (≤1%) 否
抑制剂/螯合剂 Antipain (≤100μg/ml) 否
Chymostatin (≤10μg/ml) 否
Leupeptin (≤10μg/ml) 否
PMSF (≤200μM) 否
Trypsin (≤10μg/ml) 否
EDTA(≤1mM) 否
EGTA(≤1mM) 否
其它试剂 Sucrose(250mM) 是
Glycerol(≤10%) 否

2. 试剂盒的准备工作：
a. TBA储存液的配制：称取适量TBA，用TBA配制液配制成浓度为0.37%的TBA储存液。例如18.5mg TBA用5ml TBA配制液配制，或者25mg TBA用6.76ml TBA配制液配制，最终浓度即为0.37%。TBA配制液需完全溶解后再使用，可以加热到70℃以促进溶解。TBA储存液较难溶解，需加热到70℃，并通过剧烈Vortex以促进溶解。配制好的TBA储存液室温避光保存，至少3个月内有效。
b. MDA检测工作液的配制: 根据待测定的样品数(含对照)，参考下表在临检测前新鲜配制适量的MDA检测工作液

检测次数 1次 10次 20次 50次
TBA稀释液 150μl 1500μl 3000μl 7500μl
TBA储存液 50μl 500μl 1000μl 2500μl
抗氧化剂 3μl 30μl 60μl 150μl

注意: MDA检测工作液较难溶解，可以70℃加热，并剧烈Vortex以促进溶解。也可以通过超声处理以促进溶解。配制好的MDA检测工作液必须当天使用。
c. 标准品的稀释：取适量标准品用蒸馏水稀释至1、2、5、10、20、50μM，用于后续制作标准曲线。如果样品中MDA的浓度很高，可以增加100、150和200μM的标准品浓度。
3. 样品测定:
a. 在离心管或其它适当容器内加入0.1ml匀浆液、裂解液或PBS等适当溶液作为空白对照，加入0.1ml上述不同浓度标准品用于制作标准曲线，加入0.1ml样品用于测定；随后加入0.2ml MDA检测工作液。可参考下表设置检测反应体系：

空白对照 标准品 样品
匀浆液、裂解液或PBS 0.1ml － －
标准品 － 0.1ml －
待测样品 － － 0.1ml
MDA检测工作液 0.2ml 0.2ml 0.2ml

b. 混匀后，100℃或沸水浴加热15分钟。加热时务必注意避免液体暴沸溅出。如果使用加热块(Heat block)进行加热注意用重物压紧离心管盖；如果使用沸水浴，则需使用可把盖子锁死的离心管或螺旋盖离心管，或用Parafilm封住离心管口，用针头刺一小孔。最方便和准确的加热方法是使用带有热盖并可以加热0.5ml PCR管的PCR仪。
c. 水浴冷却至室温，1000g室温离心10分钟。取200微升上清加入到96孔板中，随后用酶标仪在532nm测定吸光度。如果不方便测定532nm的吸光度，也可以测定530-540nm之间的吸光度。可以设定450nm为参考波长进行双波长测定。
d. MDA含量的计算：对于血浆、血清或尿液等样品可以直接根据标准曲线计算获得MDA的摩尔浓度，对于细胞、或组织样品，计算出样品溶液中的MDA含量后，可以通过单位重量的蛋白含量或组织重量等来表示最初样品中的MDA含量，例如μmol/mg蛋白或μmol/mg组织。
常见问题：
1. 没有检测到MDA。
可能样品中MDA浓度过低，在检测限之下。在检测组织或细胞的MDA时，请注意使用更多的组织或细胞。并注意尽量不要稀释样品。



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FREE RADICAL BIO MED 2015 Dec;89:209-19. (IF 6.17)
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Diabetes 2015 Dec;64(12):4011-22. (IF 7.72)
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Environ Pollut 2015 Dec 16;210:48-56. (IF 6.792)
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J Pharm Pharmacol 2015 Dec;67(12):1705-15. (IF 2.571)
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KIDNEY BLOOD PRESS R 2016;41(2):129-38. (IF 1.898)
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PLoS One 2016 Jan 25;11(1):e0147858. (IF 2.74)
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J Exp Bot 2016 Apr;67(9):2829-45. (IF 5.908)
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S0131S
100次
脂质氧化(MDA)检测试剂盒
S0053
共100次
GSH和GSSG检测试剂盒
S0101M
250次
总SOD活性检测试剂盒(WST-8法)
S0101S
100次
总SOD活性检测试剂盒(WST-8法)
S0033S
>100次
活性氧检测试剂盒
S0051
100次
过氧化氢酶检测试剂盒
S0043S
100-1000次
脂质过氧化检测试剂盒(BODIPY 581/591 C11)
P0013
100ml
Western及IP细胞裂解液
S0052
100次
总谷胱甘肽检测试剂盒
S0033M
>500次
活性氧检测试剂盒
S0057S
100次
谷胱甘肽过氧化物酶检测试剂盒(DTNB法)
S0109
100次
总SOD活性检测试剂盒(NBT法)