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报告基因相关

萤火虫萤光素酶报告基因检测试剂盒
产品编号: RG005
产品包装: 100次
产品价格: 398.00元
产品简介
使用说明
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产品编号 产品名称 产品包装 产品价格
RG005 萤火虫萤光素酶报告基因检测试剂盒 100次 398.00元

      碧云天生产的萤火虫萤光素酶报告基因检测试剂盒(Firefly Luciferase Reporter Gene Assay Kit),是一种以萤光素
(luciferin)为底物来检测萤火虫萤光素酶(firefly luciferase)活性的试剂盒。
      萤火虫萤光素酶是一种分子量约为61kD的蛋白,在ATP、镁离子和氧气存在的条件下,可以催化luciferin氧化成oxyluciferin。在luciferin氧化的过程中,会发出生物萤光(bioluminescence)。生物萤光可以通过化学发光仪(luminometer)或液闪测定仪进行测定。本试剂盒的检测原理参考图1。

图1. 萤火虫萤光素酶的检测原理图。

      通过萤光素和萤光素酶这一生物发光体系,可以非常灵敏、高效地检测基因的表达。通常把感兴趣基因的转录调控元件或5’启动子区克隆在luciferase的上游,或把3’-UTR区克隆在luciferase的下游等,构建成报告基因(reporter gene)质粒。然后转染细胞,用适当药物等处理细胞后裂解细胞,测定萤光素酶活性。通过萤光素酶活性的高低来判断药物处理等对目的基因的转录调控作用。
      萤火虫萤光素酶催化luciferin发光的最强发光波长为560nm(centered around 560nm)。
      本试剂盒可以测定100个样品。
包装清单:

产品编号 产品名称 包装
RG005-1 报告基因细胞裂解液 60ml
RG005-2 萤火虫萤光素酶检测试剂 10ml
说明书 1份

保存条件:
      报告基因细胞裂解液4℃保存3个月有效,-20℃保存一年有效;萤光素酶检测试剂-20℃避光保存6个月有效,-80℃避光保存一年有效。
注意事项:
      为取得最佳测定效果,在用单管的化学发光仪测定时,样品和测定试剂混合后到测定前的时间应尽量控制在相同时间内,例如30秒内;使用具有化学发光测定功能的多功能荧光酶标仪时,宜先把样品全部加好,然后统一加入萤火虫萤光素酶检测试剂。
      由于温度对酶反应有影响,所以测定时样品和试剂均需达到室温后再进行测定。
      为保证萤光素酶检测试剂的稳定性,可以采取适当分装后避光保存的方法,以避免反复冻融和长时间暴露于室温。经测试,反复冻融三次,对测定结果无明显影响。
      样品和测定试剂混合后,必须等待1-2秒,再进行测定。测定时间通常为10秒,根据情况也可以测定更长或更短时间,但是同一批样品宜使用相同的测定时间。
      为避免由于质粒转细胞时效率的差异而带来的误差,可以同时转入海肾萤光素酶(Renilla luciferase)的报告基因质粒作为内参,采用碧云天的双萤光素酶报告基因检测试剂盒(RG027/RG028)进行检测;也可以同时转入β-半乳糖苷酶(β-galactosidase,β-gal)报告基因质粒作为内参,然后采用碧云天生产的β-半乳糖苷酶报告基因检测试剂盒(RG0036)进行检测。采用本试剂盒中的报告基因细胞裂解液裂解获得的样品可以直接用于β-半乳糖苷酶报告基因检测试剂盒(RG0036)的检测。
      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. 裂解细胞:将报告基因细胞裂解液充分混匀后,按如下方式加入报告基因细胞裂解液,充分裂解细胞。
a. 对于贴壁细胞:吸尽细胞培养液后,参考下表加入适量的报告基因细胞裂解液;对于悬浮细胞:离心去上清后,参考下表加入适量报告基因细胞裂解液。
器皿类型 96孔板 48孔板 24孔板 12孔板 6孔板
报告基因细胞裂解液 (微升/孔) 100 150 200 300 500
注:如果荧光素酶的表达水平比较低,可以尝试使用更少的裂解液,例如6孔板的每孔用量可以最小为100微升。
b. 充分裂解后,10,000-15,000g离心3-5分钟,取上清用于测定。
注:细胞裂解后可立即测定萤光素酶,也可以先冻存,待以后再测定。冻存样品需融解,并达到室温后再进行测定。
2. 融解萤光素酶检测试剂,并达到室温。
3. 按仪器操作说明书开启化学发光仪或具有检测化学发光功能的多功能酶标仪,将测定间隔设为2秒,测定时间设为10秒。
4. 每个样品测定时,取样品20-100微升(如果样品量足够,请加入100微升;如果样品量不足可以适当减少用量,但同批样品的使用量宜保持一致)。
5. 加入100微升萤光素酶检测试剂,用枪打匀或用其它适当方式混匀后测定RLU(relative light unit)。以报告基因细胞裂解液为空白对照。本试剂盒的检测效果以及与同类竞争产品的检测效果比较可以参考图2。

图2. 本产品和P公司竞争产品(Competitor P)对相同样品的检测效果。实际读数会因细胞数量、转染效果、检测仪器等的不同而存在差异,图中数据仅供参考。

常见问题:
1. Luminometer和荧光分光光度计有何不同?
荧光分光光度计检测的样品本身不能发光,样品需要由特定波长的激发光激发,然后才能产生荧光并被荧光分光光度计检测。Luminometer检测的样品本身可以发光,不需要激发光进行激发。也就是说luminometer是检测化学发光(萤光)的仪器。有些型号的荧光分光光度计也具有luminometer的功能,即也可以检测化学发光。您所使用的荧光分光光度计能否用于化学发光的测定请仔细阅读该仪器的说明书。
2. 可以进行ATP化学发光检测的仪器是否就可以用于本试剂盒的检测?
是。ATP化学发光的检测原理和本试剂盒的原理相同,可以用相同的仪器测定。

相关产品:

产品编号 产品名称 包装
RG005 萤火虫萤光素酶报告基因检测试剂盒 100次
RG006 萤火虫萤光素酶报告基因检测试剂盒 1000次
RG016 海肾萤光素酶报告基因检测试剂盒 100次
RG017 海肾萤光素酶报告基因检测试剂盒 1000次
RG027 双萤光素酶报告基因检测试剂盒 100次
RG028 双萤光素酶报告基因检测试剂盒 1000次
RG0036 β-半乳糖苷酶报告基因检测试剂盒 200次
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