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信号转导> 信号小分子检测> 活性氧相关
脂质氧化(MDA)检测试剂盒
产品编号: S0131M
产品包装:500次
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价格:¥1368.00元
产品简介
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产品编号 产品名称 产品包装 产品价格
S0131S 脂质氧化(MDA)检测试剂盒 100次 366.00元
S0131M 脂质氧化(MDA)检测试剂盒 500次 1368.00元

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)
HEPES (100mM)
Phosphate (100mM)
Tris (25mM)
去垢剂 CHAPS (≤1%)
Triton X-100 (≤1%)
Tween 20 (≤1%)
抑制剂/螯合剂 Antipain (≤100μg/ml)
Chymostatin (≤10μg/ml)
Leupeptin (≤10μg/ml)
PMSF (≤200μM)
Trypsin (≤10μg/ml)
EDTA(≤1mM)
EGTA(≤1mM)
其它试剂 Sucrose(250mM)
Glycerol(≤10%)

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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