[實驗技術]西方點墨法

General Western blot

Makes 15 ml of 5x Loading Dye

3ml of 20% SDS
3.75 mL 1M Tris - pH 6.8
9 mg bromphenol blue
1.16 gm DTT (Alternatively add 2.4ml B-mercaptoethanol)
Add water to a final volume of 10.5ml
Mix the above reagents well before adding glycerol
4.5 mL Glycerol
Mix well again Store at 4 degrees


Running Buffer (1L)
Glycine 14.4 g
Tris 3.03 g
10% SDS 10 mL

Running condition
50V for 10~15 min
100V for 90 min



Transfer buffer (1 L)
Glycine 14.4 g
Tris 3.03 g
Methanol 100 mL

Transfer condition
200 mA/set for 120 min
blocking 5% skim milk / TBST

10X TBS Buffer (Tris buffer saline) (1 liter)
Tris Base [121.14] 500mM 60.5 g
Sodium Chloride (NaCl) 1.5 M 87.6 g
Add dH2O dilute to 1L pH= 7.5（以HCl滴定pH）


TBST (TBS + 0.05% Tween 20)
一抗 O/N, TBST 5 min x 6,
二抗 不超過2hr, TBST, x 6
Strip, ddH2O, microwave for 5 mins
Blocking 10% skim milk / TBST O/N 進行下一個階段的實驗