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如何选择合适的二抗

 

二抗是什么?

二抗是能和一抗结合的抗体,即抗体的抗体。主要用于检测一抗的存在。一抗特异性结合抗原的,二抗是特异性结合一抗。即一抗的Fc段或Fab片段可以充当抗原刺激机体产生抗体。也就是说,一抗主要是IgG类免疫球蛋白,因此二抗就是抗IgG抗体。
 

一抗二抗都是一种可以特异结合别的物质的基团,而且一抗可以至少结合两种其他基团(底物和二抗),如下图是直标一抗。

1.png

 

二抗的作用
 


一抗是特异性的结合目标蛋白,一抗需要发光物质或者显色基团标记才能被检测出来,否者一抗和蛋白的结合与否用肉眼是看不出来的。但是一抗往往比较特异每个都标记成本很高,通常会使用标记二抗可以和一抗结合,并带有可以被检测出的标记(如带荧光、放射性、化学发光或显色基团),所以如今的设计一般是二抗带上可检测标记,再来检测一抗,而一抗识别底物。这样,当一抗结合到底物上,就可以通过二抗检测出来。

比如ECL 显色原理:在 ECL 底物中,含有 H2O2 和鲁米诺(及其衍生物),在 HRP(辣根过氧化物酶)的作用下,发出荧光。

2.png

 

 

二抗使用要点

二抗是由一抗种属(比如兔,小鼠)的某一亚型的免疫球蛋白IgG免疫哺乳动物(一般是免疫山羊、马、驴、骡和豚鼠及小鼠等动物)产生的抗体,因此二抗只结合特定的物种特定亚型的一抗。比如,羊抗小鼠(二抗)会特异性结合小鼠单抗(一抗)而不会结合兔多抗或大鼠单抗,所以二抗和一抗要匹配上。

 

 

二抗的耦联标记

实验方法不同,二抗耦联的基团就会不同,通常主要有酶(辣根过氧化酶HRP和碱性磷酸酶AP),荧光基团(FITC、 Rhodamine、PE、Dylight、Alexa Fluor 、cy3等)、生物素、金颗粒、。一般做WB、 ELISA、 IHC等实验都是推荐HRP标记的二抗,IHC推荐生物素标记的二抗,做IF、 FC等都是荧光如Alexa Fluor 488、FITC、PE标记的抗体。
 

 

如何选择合适的二抗呢

a.免疫印记(WB)

图片1.png

 

b.免疫组化(IHC/ICC)

图片2.png


c.免疫荧光(IHF/ICF)

图片3.png

 

d.免疫沉淀免疫共沉淀

 

1652154598388928.png


特殊二抗

图片5.png

 

推荐产品信息
 

 

标记基团

产品货号

产品名称

无标记

34850ES

Goat Anti-Rabbit lgG(H+L)

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YSFluor™ 594 Goat Anti-Bovine IgG (H+L)

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YSFluor™594 Rabbit Anti-Goat IgG (H+L)

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YSFluor™ 594 Rabbit Anti-Sheep IgG (H+L)

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YSFluor™594 Rabbit Anti-Mouse IgG (H+L)

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YSFluor™594 Rabbit Anti-Chicken IgY (H+L)

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YSFluor™ 594 Donkey Anti-Mouse IgG (H+L)

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YSFluor™ 594 Donkey Anti-Goat IgG (H+L)

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Cy3-AffiniPure Goat Anti-Mouse IgG (H+L)

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YSFluor™ 647 Rabbit Anti-Goat IgG (H+L)

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YSFluor™647 Rabbit Anti-Sheep IgG (H+L)

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YSFluor™ 647 Rabbit Anti-Mouse IgG (H+L)

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YSFluor™647 Rabbit Anti-Chicken IgY (H+L)

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YSFluor™ 647 Donkey Anti-Mouse IgG (H+L)

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YSFluor™ 647 Donkey Anti-Rabbit IgG (H+L)

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YSFluor™ 647 Donkey Anti-Rat IgG (H+L)

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YSFluor™647 Donkey Anti-Human IgG (H+L)

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