Techniques如何选择合适的二抗
如何选择合适的二抗
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。
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。
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。
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。
a.免疫印记（WB）
b.免疫组化（IHC/ICC）
c.免疫荧光（IHF/ICF)
d.免疫沉淀免疫共沉淀
特殊二抗
抗体的应用选购指南
标记基团 | 产品货号 | 产品名称 |
无标记 | Goat Anti-Rabbit lgG(H+L) | |
Goat Anti-Mosue lgG(H+L) | ||
Donkey Anti-Human IgG(H+L) | ||
HRP | Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) | |
Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) | ||
Peroxidase AffiniPure Goat Anti-Rat IgG (H+L) | ||
Peroxidase AffiniPure Goat Anti-Guinea Pig IgG(H+L) | ||
Horseradish Peroxidase Goat Anti-Human IgG (H+L) | ||
Peroxidase AffiniPure Rabbit Anti-Goat IgG (H+L) | ||
Peroxidase AffiniPure Rabbit Anti-Sheep IgG (H+L) | ||
Peroxidase AffiniPure Donkey Anti-Rabbit IgG (H+L) | ||
Peroxidase AffiniPure Donkey Anti-Mouse IgG (H+L) | ||
Peroxidase AffiniPure Donkey Anti-Goat IgG (H+L) | ||
Biotin | Biotin-SP AffiniPure Goat Anti-Rabbit IgG (H+L) | |
Biotin-SP AffiniPure Goat Anti-Mouse IgG (H+L) | ||
Biotin-SP AffiniPure Goat Anti-Rat IgG (H+L) | ||
Biotin-SP AffiniPure Donkey Anti-Goat IgG (H+L) | ||
绿色荧光 | YSFluor™ 488 Goat Anti-Rabbit IgG (H+L) | |
YSFluor™ 488 Goat Anti-Human IgG (H+L) | ||
YSFluor™488 Goat Anti-Mouse IgG (H+L) | ||
YSFluor™ 488 Goat Anti-Bovine IgG (H+L) | ||
YSFluor™ 488 Goat Anti-Guinea Pig IgG (H+L) | ||
YSFluor™488 Rabbit Anti-Goat IgG (H+L) | ||
YSFluor™ 488 Rabbit Anti-Mouse IgG (H+L) | ||
YSFluor™488 Donkey Anti-Sheep IgG (H+L) | ||
YSFluor™488 Donkey Anti-Mouse IgG (H+L) | ||
YSFluor™488 Donkey Anti-Rabbit IgG (H+L) | ||
YSFluor™ 488 Donkey Anti-Goat IgG (H+L) | ||
YSFluor™ 488 Donkey Anti-Rat IgG (H+L) | ||
YSFluor™488 Donkey Anti-Guinea Pig IgG (H+L) | ||
YSFluor™488 Donkey Anti-Chicken IgG (H+L) | ||
YSFluor™488 Rabbit Anti-Chicken IgY(H+L) | ||
YSFluor™488 Donkey Anti-Human IgG (H+L) | ||
FITC AffiniPure Goat Anti-Human IgG(H+L) | ||
FITC-AffiniPure Goat Anti-Rabbit IgG (H+L) | ||
FITC-AffiniPure Goat Anti-Mouse IgG (H+L) | ||
FITC-AffiniPure Goat Anti-Rat IgG (H+L) | ||
FITC-AffiniPure Rabbit Anti-Goat IgG (H+L) | ||
FITC-AffiniPure Rabbit Anti-Sheep IgG (H+L) | ||
罗丹明 | Rhodamine (TRITC) Goat Anti-Rabbit IgG (H+L) | |
Rhodamine (TRITC) Goat Anti-Mouse IgG (H+L) | ||
Rhodamine(TRITC) AffiniPure Rabbit Anti-Goat IgG (H+L) | ||
Rhodamine Red-X (RRX) Goat Anti-Mouse IgG (H+L) | ||
橙色荧光 | YSFluor™594 AffiniPure Goat Anti-Rabbit IgG (H+L) | |
YSFluor™ 594 Goat Anti-Mouse IgG (H+L) | ||
YSFluor™ 594 Goat Anti-Human IgG (H+L) | ||
YSFluor™594 Goat Anti-Guinea Pig IgG (H+L) | ||
YSFluor™ 594 Goat Anti-Bovine IgG (H+L) | ||
YSFluor™594 Rabbit Anti-Goat IgG (H+L) | ||
YSFluor™ 594 Rabbit Anti-Sheep IgG (H+L) | ||
YSFluor™594 Rabbit Anti-Mouse IgG (H+L) | ||
YSFluor™594 Rabbit Anti-Chicken IgY (H+L) | ||
YSFluor™ 594 Donkey Anti-Mouse IgG (H+L) | ||
YSFluor™594 Donkey Anti-Rabbit IgG (H+L) | ||
YSFluor™ 594 Donkey Anti-Goat IgG (H+L) | ||
YSFluor™ 594 Donkey Anti-Rat IgG (H+L) | ||
YSFluor™594 Donkey Anti-Guinea Pig IgG (H+L) | ||
YSFluor™594 Donkey Anti-Chicken IgG (H+L) | ||
YSFluor™594 Donkey Anti-Human IgG (H+L) | ||
YSFluor™594 Donkey Anti-Sheep IgG (H+L) | ||
Cy3-AffiniPure Goat Anti-Mouse IgG (H+L) | ||
Cy3-AffiniPure Goat Anti-Rat IgG (H+L) | ||
Cy3-AffiniPure Rabbit Anti-Goat IgG (H+L) | ||
Cy3-AffiniPure Rabbit Anti-Sheep IgG (H+L) | ||
Cy3-AffiniPure Donkey Anti-Goat IgG(H+L) | ||
红色荧光 | YSFluor™647 Goat Anti-Rabbit IgG (H+L) | |
YSFluor™ 647 Goat Anti-Mouse IgG (H+L) | ||
YSFluor™647 Goat Anti-Human IgG (H+L) | ||
YSFluor™ 647 Goat Anti-Bovine IgG (H+L) | ||
YSFluor™ 647 Goat Anti-Guinea Pig IgG (H+L) | ||
YSFluor™ 647 Rabbit Anti-Goat IgG (H+L) | ||
YSFluor™647 Rabbit Anti-Sheep IgG (H+L) | ||
YSFluor™ 647 Rabbit Anti-Mouse IgG (H+L) | ||
YSFluor™647 Rabbit Anti-Chicken IgY (H+L) | ||
YSFluor™ 647 Donkey Anti-Mouse IgG (H+L) | ||
YSFluor™ 647 Donkey Anti-Rabbit IgG (H+L) | ||
YSFluor™ 647 Donkey Anti-Rat IgG (H+L) | ||
YSFluor™647 Donkey Anti-Human IgG (H+L) | ||
YSFluor™647 Donkey Anti-Sheep IgG (H+L) |
文献引用
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