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NADP+/NADPH检测试剂盒(WST-8法)

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  • 碧云天(beyotime)已认证
  • S0179
  • 2026年05月29日
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    碧云天的NADP+/NADPH检测试剂盒(WST-8法) (NADP+/NADPH Assay Kit with WST-8)是一种基于WST-8的显色反应,通过比色法来检测细胞、组织或其它样品中NADP+ (氧化型辅酶II)和NADPH (还原型辅酶II)各自的量、比值和总量的检测试剂盒。
    NADP+/NADPH以及NAD+/NADH的传统检测方法是检测NADPH或者NADH在340nm处吸收波长的变化,该方法灵敏度较低并易受样品中有类似紫外吸收物质的干扰,并且在紫外检测过程中通常需要加大检测样品量以弥补NADPH在340nm处吸光度过小的不足,因此该检测方法具有很大的局限性。
    WST-8是MTT的一种升级替代产品,和MTT或其它MTT类似产品如XTT、MTS等相比有明显的优点。首先,MTT被一些脱氢酶还原生成的formazan不是水溶性的,需要有特定的溶液来溶解;而WST-8和XTT、MTS产生的formazan都是水溶性的,可以省去后续的溶解步骤。其次,WST-8产生的formazan比XTT和MTS产生的formazan更易溶解。再次,WST-8比XTT和MTS更加稳定,使实验结果更加稳定。另外,WST-8和MTT、XTT等相比,线性范围更宽,灵敏度更高。
    WST-8和WST-1相比,检测灵敏度更高,更易溶解,并且更加稳定。
    本试剂盒使用便捷,无需分离纯化细胞、组织或其它样品中的NADP+和NADPH,且特异性检测NADP+和NADPH,而不检测NAD+和NADH。本试剂盒可以检测含量低至0.05μM (2.5pmol)的NADP+或NADPH,在0.05μM (2.5pmol)至6μM (300pmol)之间呈现良好的线性关系。
    NADP (Nicotinamide adenine dinucleotide phosphate,烟酰胺腺嘌呤二核苷酸磷酸)是很多氧化还原反应的辅酶,包括NADP+ (氧化型)和NADPH (还原型)两种形式。NADP+也参与到生物合成反应中,比如脂质和核酸的合成。在动物细胞中,戊糖磷酸途径(pentose phosphate pathway,PPP)的氧化阶段是NADPH最主要的来源。
    本试剂盒可检测样品中的NADP+、NADPH以及它们的比值,具体原理如下:

    a. 测定NADP+/NADPH的总量:葡萄糖-6-磷酸(glucose-6-phosphate, G6P)在葡萄糖-6-磷酸脱氢酶(glucose-6-phosphate dehydrogenase, G6PDH)的作用下氧化生成6-磷酸葡萄糖酸内酯(6-phosphogluconate,6-PG),在这一反应过程中NADP+被还原为NADPH;生成的NADPH在电子耦合试剂1-mPMS (1-Methoxy-5-methylphenazinium Methyl Sulfate)的作用下将WST-8还原生成橙黄色的formazan,在450nm左右检测有最大吸收峰。反应体系中生成的formazan与样品中总的NADP+/NADPH的总量呈比例关系。WST-8法检测NADP+和NADPH总量的原理参考图1。
    b. 单独测定NADPH的量:60℃水浴加热30分钟后,样品中NADP+会分解而只保留NADPH。NADPH将WST-8还原成formazan,通过比色法确定反应生成的formazan的量,最终可以确定样品中NADPH的量。
    c. 测定NADP+以及NADP+/NADPH比值:根据前两步检测获得的NADP+和NADPH总量以及NADPH的量,即可得到样品中NADP+的量以及NADP+/NADPH的比值。

     

    产品细节图片1
    图1.NADP+/NADPH检测试剂盒(WST-8法) (S0179)的工作原理图。

    本试剂盒适用于检测细胞、组织以及其它适当样品中的NADP+和NADPH各自的量、比值和总量。
    本试剂盒提供的NADP+/NADPH提取液有一定的通用性。使用本试剂盒中的NADP+/NADPH提取液提取获得的细胞或组织样品,也可以用于碧云天生产的NAD+/NADH检测试剂盒(WST-8法) (S0175)的检测。
    当仅检测NADP+和NADPH的总量或者样品中的NADPH的量时,一个本试剂盒可以进行100次检测;当检测NADP+或者NADP+/NADPH的比值时,一个本试剂盒可以进行50次检测。
    包装清单:

    产品编号 产品名称 包装
    S0179-1 G6PDH 220μl
    S0179-2 显色液 1.1ml
    S0179-3 NADPH 5mg
    S0179-4 NADP+/NADPH提取液 50ml
    S0179-5 反应缓冲液 11ml
    说明书 1份

    保存条件:
    -20℃保存,一年有效。显色液(S0179-2)和NADPH (S0179-3)须-20℃避光保存。NADPH配制成溶液后,须适当分装后-80℃保存。所有试剂避免反复冻融。
    注意事项:
    本试剂盒中的所有试剂均需要冷冻保存,请严格按照保存条件进行保存。如果不是一次用完,为避免反复冻融导致产品失效,请适当分装后保存。
    NADPH不太稳定,取出NADPH后请尽快使用。如果发现标准曲线不理想,很有可能是标准品发生了降解。
    由于NADP+/NADPH提取液本身比较粘稠,以该提取液作为稀释液时,无论对标准品还是样品进行稀释,在稀释过程中务必保证稀释均匀,否则易造成实验数据产生较大波动。
    细胞或组织样品的提取,请严格按照每100万个细胞或10mg组织加入200μl提取液的比例加入NADP+/NADPH提取液。如果提取液用量过少,得到的样品提取液可能会出现比较粘稠、不易吸取的情况,影响实验操作的便利性,进而影响检测结果的准确性。推荐使用增强型NADP+/NADPH检测试剂盒(WST-8法) (S0180),可有效解决在提取细胞和组织样品时可能出现的粘稠问题。
    在样品加样和混匀过程中,须尽量避免产生气泡,以免影响最终的吸光度测定。
    如果不能非常严格地控制反应温度和反应时间,每次检测都需要设置标准曲线。
    如果样品溶液中NADP+和NADPH浓度过高或过低,不在试剂盒的线性检测范围内时,可适当调整样品或者提取液的用量。
    由于NADP+和NADPH很不稳定,在冻存过程中较易降解,所以宜尽量使用新鲜样品进行检测。如果需要进行样品中NADP+以及NADP+/NADPH比值的测定,在样品加热30分钟以分解NADP+的过程中,可以先进行样品中NADP+和NADPH的总量的测定,即将NADP+和NADPH的总量的测定和单独NADPH的测定分开进行,尽量减少样品在等待过程中因为可能的降解导致的误差。本试剂盒中的所有试剂均需要冷冻保存,请严格按照保存条件进行保存。如果不是一次用完,为避免反复冻融导致产品失效,请适当分装后保存。
    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。
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