實驗概要

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1.目的??檢測小分子蛋白抗體

2.適用范圍??單克隆抗體和多克隆抗體

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實驗原理

將經(jīng)電泳分離的抗原轉(zhuǎn)印到膜上作為固相,利用酶標(biāo)記的抗抗體以檢測已與固相抗原結(jié)合的受檢抗體

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主要試劑

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試劑配制

4.1.1??40%?Bis-acrylamide?with?2.6%?C?（Acrylamide:Bis,?38.95:1.05）

??????丙烯酰胺?????????77.9g

??????雙甲叉丙烯酰胺???2.1g

??????Total?????????????200ml

4.1.2??30%膠溶液:

0.8????雙甲叉丙烯酰胺????0.8g

29.2%?丙烯酰胺??????????29.2g

????????total?????????????100ml

4.1.3??3M?TrisHCl?,?pH?8.45

??????36.3g????Trisbase??

??????Total????100ml

4.1.4???WG?(?water? ?Glycerol):???

???????Water:?????58ml

???????Glycerol:???36ml

???????Total:??????94ml

4.1.5????10x?Electrophoresis?Buffer?stock?pH8.3?（to?make?the?working?buffer,?just?dilute?10?times.?Do?not?adjust?pH?,?pH?should?be?8.3.）

Trisbase???????60.5g

Tricine????????89.5g

SDS??????????5g

??????????total??????????500ml

4.1.6???10%?SDS

????????SDS????10g

????????Total????100ml

4.1.7????2x?Laemmli?Stacking?Buffer?pH6.8:

0.25M?Tris???????15.14g

0.2%?SDS????????1g?or?5ml?20%?SDS

??????????total?????????500ml

4.1.8??5x?Stop?Solution?(Sample?buffer)?pH6.7:

Stock:????????????????????????????????????to?use:

20%?Glycerol??????100ml??????????????????take?9.5ml?stock????????

10%?SDS?????????50g?or?250ml?20%?SDS????add?BME?0.5ml

250mM?Tris???????15.14g??????????????????bromphenol?blue?or?

??????????total???????????475.00ml????????????????pyronine?4?to?taste

4.1.9??transfer?buffer??????????????????????

48mM?Tris?base??????5.81g

39mM?Glycine???????2.93g

0.037%?SDS?????????0.375g?or?1.875ml?of?20%?SDS

40%?MeOH??????????400ml

total???????????1000ml

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4.1.10??TBST

10mM?Tris-HCl,?pH7.4

100mM?NaCl

0.2%?Tween-20

?????????Total??1000ml

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