Tricine SDS-PAGE实用方案
Tricine-SDS-PAGE是用于分离分子量在1-10kD的肽类用的。
一、 试剂配制：
1． Low Bis acrylamide（49.5% T, 3% C）
Acylamide 48.0g
Bis 1.5g
Water make up to 100ml
2. High Bis acrylamide (49.5% T, 6% C)
Acrylamide 46.5g
Bis 3.0g
Water make up to100ml
3. Gel Buffer (3M Tris/cl, PH8.45, 0.3% SDS)
SDS 0.3g
Tris 36.4g
Water make up to 100ml
PH to 8.45 with HCL
注: 3M Tris 配制时不容易溶解,因此配制时我配制了2M Tris,配方如下:
Tris 36.4g
SDS 0.3g
Water make up to 150ml
4. Anode (Lower) buffer 阳极缓冲液 (0.2M Tris/cl, PH8.9)
Tris 12.11g
Water make up to 500ml
PH to 8.9 with HCL
5. Cathode (Upper) buffer 阴极缓冲液 (0.1M Tris/cl, 0.1M Tricine, 0.1% SDS, PH 8.25)
Tris 6.06g
Tricine 8.96g
SDS 0.5g
Water make up to 500ml
注:不用调PH值
6. 4×Tricine SDS Sample buffer 4×上样缓冲液
(Final concentration: 0.05M Tris/cl, 4% SDS, 12% glycerol, 200mM DTT, 0.01% Commasie G 250)
8×Tris.cl/SDS PH 6.8 2ml
Glycerol 4.8ml
SDS 1.6g
Commasie Blue G 250 4mg
Water make up to 10ml
注:8×Tris.cl/SDS PH 6.8配方
Tris 6.05g
SDS 0.4g
Water make up to 100ml
PH to 6.8 with HCL
二、 胶的配制
1. 16.5% T 6% C separating gel 30ml
49.5% T 6% C 10ml
Gel buffer 15ml
Glycerol 3.2ml
Water 1.8ml
2. 10% T 3% C spacer gel 30ml
49.5% T 3% C 6.1ml
Gel buffer 15ml
Water 8.9ml
3. 4% T 3% C stacking gel 12.5ml
49.5% T 3% C 1ml
Gel buffer 4.65ml
Water 6.85ml
注: 用Bio-Rad系统, 0.75mm的胶,separating gel 配3ml, 加2.5ml; Spacer gel 配1ml,加0.7ml; Stacking gel 配1.25ml. 灌胶前临时加10%AP（过硫酸铵）和TEMED.
胶配制的通用配方:
三、电泳参数及染色脱色
1. 用Bio-Rad mini 电泳装置进行电泳. 30V 电泳1hr, 100V至电泳结束
2. 固定, 染色及脱色: 小分子量肽类在SDS-PAGE胶中容易扩散,因此电泳结束后, 有时需要固定20min (固定液: 0.5% 戊二醛,
30% 乙醇), 再进行染色20-30min (染色液: 50% 甲醇, 10% 乙醇, 0.2% G-250), 在脱色液 (45% 甲醇,
10% 冰醋酸)脱色20-30min; 脱色完毕,把胶放在水中或10%甘油中。
