化学发光法EMSA试剂盒
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产品编号 GS009
数量
¥ 1662.00
产品包装:
100次
产品简介
使用说明
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化学发光法EMSA试剂盒(Chemiluminescent EMSA Kit)是一种通过Streptavidin-HRP及后续的BeyoECL Moon试剂来实现化学发光检测Biotin标记的EMSA探针的检测试剂盒。同时本试剂盒也提供了EMSA检测所需的结合缓冲液和上样缓冲液,及一些关键的相关试剂,可以实现非同位素的EMSA检测。
本试剂盒采用了高质量的Streptavidin-HRP Conjugate,HRP和Streptavidin共价交联的比例大于3,这样比采用
Streptavidin和Biotin-HRP conjugate两种试剂进行检测要更方便,并且灵敏度更高。
本试剂盒采用了非特异性结合比avidin更低的strepatavidin,使检测结果背景更低灵敏度更高。
本试剂盒和碧云天的各种生物素标记EMSA探针可以配套使用。
本试剂盒没有提供生物素探针标记相关的试剂,其它特定的生物素标记EMSA探针的制备可以使用碧云天的EMSA探针生物素标记试剂盒(GS008)
EMSA/Gel-Shift结合缓冲液(5X)中含有poly(dI-dC)等有效成分。其中poly(dI-dC)的浓度经过优化,可以很好的消除蛋白和标记探针间的非特异性结合,同时又不会减弱目的转录因子和标记探针间的结合。
本试剂盒可以用于100个蛋白和探针的结合反应,并足够检测至少10块有生物素标记EMSA探针的膜。
包装清单:

产品编号 产品名称 包装
GS009-1 EMSA/Gel-Shift结合缓冲液(5X) 200μl
GS009-2 EMSA/Gel-Shift上样缓冲液(无色,10X) 200μl
GS009-3 EMSA/Gel-Shift上样缓冲液(蓝色,10X) 200μl
GS009-4 BeyoECL Moon A液 55ml
GS009-5 BeyoECL Moon B液 55ml
GS009-6 Streptavidin-HRP Conjugate 100μl
GS009-7 封闭液 380ml
GS009-8 洗涤液(5X) 250ml
GS009-9 检测平衡液 250ml
说明书 1份

保存条件:
GS009-1至GS009-3和GS009-6在-20℃保存,其余可4℃保存。如果长期不用,整个试剂盒可-20℃保存,-20℃可以保存更长时间。
注意事项:
需自备带正电荷尼龙膜,以及凝胶电泳时所需的相关试剂。带正电荷尼龙膜(FFN10/FFN11/FFN13/FFN15)可以向碧云天订购。
如果需要使用更多的封闭液或洗涤液,可另外单独订购GS009B封闭液GS009W洗涤液(5X)
BeyoECL Moon A液和B液均对人体有害,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。
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. 探针的标记:
可以直接选购碧云天的生物素标记EMSA探针,或使用碧云天生 产的EMSA探针生物素标记试剂盒 (GS008)或其它合适的试剂盒进行EMSA探针的生物素标记。
2. 探针的纯化和检测:
碧云天的各种生物素标记EMSA探针都已经过纯化,可以直接使用;对于使用碧云天的EMSA探针生物素标记试剂盒(GS008)等试剂盒标记的EMSA探针,通常 为实验简便起见,可以不必纯化标记好的探针。有些时候,纯化后的探针会改善EMSA的电泳结果。详细的探针纯化和探针的标记效率的检测请参考EMSA探针生物素标记试剂盒的相关说明。
3. EMSA胶的配制:
a.准备好倒胶的模具。可以使用常规的制备蛋白电泳胶的模具(例如BioRad的常规用于蛋白电泳的制胶装置),或其它适当的模具。最好选择可以灌制较薄 胶的模具,以便于干胶等后续操作。为得到更好的结果,可以选择可灌制较大EMSA胶的模具。制胶前必须把制胶模具冲洗干净,需特别注意不能有SDS残留。
b.按照如下配方配制20ml 4%的聚丙烯酰胺凝胶(注意:使用29:1等不同比例的Acr/Bis对结果影响不大)。

TBE buffer(10X) 1ml
重蒸水 16.2ml
39:1 acrylamide/bisacrylamide(40%,w/v) 2ml
80% 甘油 625μl
10% 过硫酸铵(ammonium persulfate) 150μl
TEMED 10μl

c.按照上述顺序依次加入各种试剂,加入TEMED前先混匀,加入TEMED后立即混匀,并马上加入到制胶的模具中。避免产生气泡,并加上梳 齿。如果发现非常容易形成气泡,可以把一块制胶的玻璃板进行硅烷化处理。
4. EMSA结合反应:
a.如下设置EMSA结合反应:

阴性对照反应:
Nuclease-Free Water 7μl
EMSA/Gel-Shift结合缓冲液(5X) 2μl
细胞核蛋白或纯化的转录因子 0μl
标记好的探针 1μl
总体积 10μl
样品反应:
Nuclease-Free Water 5μl
EMSA/Gel-Shift结合缓冲液(5X) 2μl
细胞核蛋白或纯化的转录因子 2μl
标记好的探针 1μl
总体积 10μl
探针冷竞争反应:
Nuclease-Free Water 4μl
EMSA/Gel-Shift结合缓冲液(5X) 2μl
细胞核蛋白或纯化的转录因子 2μl
未标记的探针 1μl
标记好的探针 1μl
总体积 10μl
突变探针的冷竞争反应:
Nuclease-Free Water 4μl
EMSA/Gel-Shift结合缓冲液(5X) 2μl
细胞核蛋白或纯化的转录因子 2μl
未标记的突变探针 1μl
标记好的探针 1μl
总体积 10μl
Super-shift反应:
Nuclease-Free Water 4μl
EMSA/Gel-Shift结合缓冲液(5X) 2μl
细胞核蛋白或纯化的转录因子 2μl
目的蛋白特异抗体 1μl
标记好的探针 1μl
总体积 10μl

b.按照上述顺序依次加入各种试剂,在加入标记好的探针前先混匀,并且室温(20-25℃)放置10分钟,从而消除可能发生的探针和蛋白的 非特异性结合,或者让冷探针优先反应。然后加入标记好的探针,混匀,室温(20-25℃)放置20分钟。
c.加入1μl EMSA/Gel-Shift上样缓冲液(无色,10X),混 匀后立即上样。注意:有些时候溴 酚蓝会影响蛋白和DNA的结合,建议尽量使用无色的EMSA/Gel-Shift上样缓冲液。如果对于使用无色上样缓冲液在上样时感觉到无法上样,可以在无色上样缓冲液里面添加极少量的蓝色的上样缓冲液,至可以观察到蓝颜色即可。
5.电泳:
a.用0.5XTBE作为电泳液。按照10V/厘米的电压预电泳10分钟。预电泳的时候如果有空余的上样孔,可以加入少量稀释 好的1X的EMSA上样缓冲液(蓝色),以观察电压是否正常进行。
b.把混合了上样缓冲液的样品加入到上样孔内。在多余的某个上样孔内加入10μl稀释好的1X的EMSA/Gel-Shift上样缓冲液(蓝色),用于观察电泳进行的情况。
c.按照10V/厘米的电压电泳。确保胶的温度不超过30℃,如果温度升高,需要适当降低电压。电泳至EMSA/Gel-Shift上样缓冲液中的蓝色染料溴酚蓝至胶的下缘1/4处,停止电泳。
6. 转膜:
a.取一和EMSA胶大小相近或略大的尼龙膜,剪角做好标记,用0.5XTBE浸泡至少10分钟。尼龙膜自始至终仅能使用镊子夹取,并且仅可夹取不可能接触样品的边角处。
b.取两片和尼龙膜大小相近或略大的滤纸,用0.5XTBE浸湿。
c.把浸泡过的尼龙膜放置在一片浸湿的滤纸上,注意避免尼龙膜和滤纸间产生气泡。
d.非常小心地取出EMSA胶放置到尼龙膜上,注意确保胶和膜之间没有气泡。
e.再把另外一片浸湿的滤纸放置到EMSA胶上,注意确保滤纸和胶之间没有气泡。
f.采用Western时所使用的湿法电转膜装置或其它类似的电转膜装置,以0.5XTBE为转膜液,把EMSA胶上的探针、蛋白以及探针和蛋白的复合物等转移到尼龙膜上。对于大小约为10x8x0.1cm的EMSA胶,用BioRad的常用的Western转膜装置,电转时可以设置为380mA(约100V)转膜30-60分钟。如果胶较厚,则需适当延长转膜时间。转膜时需保持转膜液的温度较低,通常可以把电转槽置于4℃冷库或置于冰浴或冰水浴中进行电转,这样可以确保低温。具体的电转膜方法请参考电转膜装置的使用说明。
g.转膜完毕后,小心取出尼龙膜,样品面向上,放置在一干燥的滤纸上,轻轻吸掉下表面明显的液体。立即进入下一步的交联步骤,不可使膜干掉。
7. 交联:
a.用紫外交联仪(UV-light cross-linker)选择254nm紫外波长,120mJ/cm2,交联45-60秒。如果没有紫外交联仪可以使用普通的手提式紫外灯(例如碧云天的手提紫外检测仪(EUV002)),距离膜5-10厘米左右照射3-10分钟。也可以使用超净工作台内的紫外灯,距离膜5-10厘米左右照射3-15分钟。最佳的交联时间可以使用标准品自行摸索。
b.交联完毕后,可以直接进入下一步检测;也可以用保鲜膜包裹后在室温干燥处存放3-5天,然后再进入下一步检测。如果检测结果发现交联效果不佳,甚至连free probe的条带都非常微弱,可以考虑在膜干燥后参考步骤A的条件再交联一次,以进一步改善交联效果。
8. 化学发光法检测生物素标记的探针:
a.37-50℃水浴溶解封闭液和洗涤液。注意: 封闭液和洗涤液必须完全溶解后方可使用,封闭液和洗涤液可以在室温至50℃之间使用,但必须确保这两种溶液中均无沉淀产生,在冬天需特别注意。
b.取一合适的容器加入15ml封闭液,再放入交联过的含有样品的尼龙膜。在侧摆摇床或水平摇床上缓慢摇动15分钟。
c.取7.5μl Streptavidin-HRP Conjugate加入到15ml封闭液中(1:2000稀释),混匀备用。
d.去除用于尼龙膜封闭的封闭液,加入上一步中配制的15ml含有Streptavidin-HRP Conjugate的封闭液。在侧摆摇床或水平摇床上缓慢摇动15分钟。
e.取25ml洗涤液(5X),加入100ml重蒸水或Milli-Q级纯水,混匀配制成125ml洗涤液。
f.将尼龙膜转移至另一装有15-20ml洗涤液的容器内,漂洗1分钟。
g.去除洗涤液,加入15-20ml洗涤液,在侧摆摇床或水平摇床缓慢上洗涤5分钟。
h.重复步骤G 三次(共洗涤四次),每次洗涤时间都约为5分钟。
i.将尼龙膜转移至另一装有20-25ml检测平衡液的容器内,在侧摆摇床或水平摇床上缓慢摇动5分钟。
j.取5ml BeyoECL Moon A液和5ml BeyoECL Moon B液混匀,配制成BeyoECL Moon工作液。
注意:BeyoECL Moon工作液必须现配现用。说明:从本步骤起操作方法和注意事项同Western实验的荧光检测。
k.取出尼龙膜,用吸水纸吸去过多液体。立即将膜的样品面向上,放置到处于水平桌面上的洁净容器内或保鲜膜上。
l.在尼龙膜的表面小心加上步骤J配制好的共10ml BeyoECL Moon工作液,使工作液完全覆盖尼龙膜。室温放置2-3分钟。
m.取出尼龙膜,用吸水纸吸去过多液体。将尼龙膜放在两片保鲜膜或其它适当的透光薄膜中间,并固定于压片暗盒(也称片夹)内。
n.用X光片压片1-5分钟。可以先压片1分钟,立即显影定影,然后根据结果再调整压片时间;也可以直接分别压片30秒、1、3、5分钟或更长时间,然后一起显影定影观察结果。

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