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WB胶及试剂配制

1.1 40% Bis-acrylamide with

2.6% C (Acrylamide:Bis, 38.95:1.05)

acrylamide 77.9g

Bis-acry 2.1g

Total 200ml

1.2 30%胶溶液:

Bis-acry 0.8g

acrylamide 29.2g

total 100ml

1.3 3M TrisHCl , pH 8.45

36.3g Trisbase

Total 100ml

1.4 WG ( water + Glycerol):

Water: 58ml

Glycerol: 36ml

Total: 94ml

1.5 10x Electrophoresis Buffer stock pH8.3

Trisbase 60.5g

Tricine 89.5g

SDS 5g

total 500ml

1.6 10% SDS

SDS 10g

Total 100ml

1.7 2x Laemmli Stacking Buffer pH6.8:

0.25M Tris 15.14g

0.2% SDS 1g or 5ml 20% SDS

total 500ml

1.8 transfer buffer

25mM Tris 3g

192mM glycine 14.4g

40% methanol 400ml

Total 1000ml

1.9 TBST

10mM Tris-HCl, pH7.4

100mM NaCl

0.2% Tween-20

Total 1000ml

WB胶及试剂配制

WB胶及试剂配制

WB胶及试剂配制

All recipes are designed for 1.5mm gel

1.1 做分离胶,加水封胶,待其凝固。

1.2 去除封胶水,做间隙胶,加水封胶,待其凝固。

1.3 去除封胶水,做浓缩胶,插梳子。

1.4 待浓缩胶凝固后,拔掉梳子,并将架子转移到电泳槽中,先在中部加入1x Electrophoresis Buffer pH8.3,点样。

1.5 调节电压到60v,开始电泳,一小时后电压升至120v。

1.6 溴酚蓝开始跑出最下端时停止电泳

1.7 取胶,湿法转移,一张膜20v过夜,2张膜串联30v过夜

1.8 封闭45min,一抗37度孵育90min,然后洗涤5min*5次,2抗孵育45min,洗涤8min*4次。曝光显影。