小分子蛋白电泳技术分享.doc

TricineSDS-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、 试剂配配制：1 Low Bis acrylamide（49.5% T, 3% C）Acylamide 48.0gBis 1.5gWater make up to 100ml2. High Bis acrylamide (49.5% T, 6% C)Acrylamide 46.5gBis 3.0gWater make up to100ml3. Gel Buffer (3M Tris/cl, PH8.45, 0.3% SDS)SDS 0.3gTris 36.4gWater make up to 100mlPH 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 36.4gSDS 0.3gWater make up to 150ml4. Anode (Lower) buffer 阳极缓冲液 (0.2M Tris/cl, PH8.9)Tris 12.11gWater make up to 500mlPH to 8.9 with HCL5. Cathode (Upper) buffer 阴极缓冲液 (0.1M Tris/cl, 0.1M Tricine, 0.1% SDS, PH 8.25)Tris 6.06gTricine 8.96gSDS 0.5gWater make up to 500ml注:不用调PH值6. 4Tricine 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)8Tris.cl/SDS PH 6.8 2mlGlycerol 4.8mlSDS 1.6gCommasie Blue G 250 4mgWater make up to 10ml注:8Tris.cl/SDS PH 6.8配方Tris 6.05gSDS 0.4gWater make up to 100mlPH to 6.8 with HCL二、 胶的配制1. 16.5% T 6% C separating gel 30ml49.5% T 6% C 10mlGel buffer 15mlGlycerol 3.2mlWater 1.8ml2. 10% T 3% C spacer gel 30ml49.5% T 3% C 6.1mlGel buffer 15mlWater 8.9ml3. 4% T 3% C stacking gel 12.5ml49.5% T 3% C 1mlGel buffer 4.65mlWater 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.四、电泳示例照片小分子该选择用tricine-SDS-PAGE. protocol上写着 the common role of glycerol in SDS gels is to increase the density of solutions and to facilitate gel casting; it has no obious effect on protein separation I tried 25% Tricine gel for 3KD peptide, looks good. Good Luck! 哦，我自己经常做2x多的分子，用的是15%的gel，效果很好的。1xKD的用16%的PAGE就应该可以了。 你可以试一试Tricine-SDS-PAGE，16%的分离胶，6%的浓缩胶。分离胶单体母液：46.5g丙烯酰胺+3g双叉丙烯酰胺，双蒸水溶解定容到100ml凝胶浓度49.5%，交联度6%浓缩胶单体母液：48g丙烯酰胺+1.5g双叉丙烯酰胺，双蒸水溶解定容到100ml凝胶浓度49.5%，交联度3%配方：分离胶 母液3.3ml+凝胶缓冲液3.3ml+30%甘油2.4ml+双蒸水1.0ml+10%AP45l+TEMED4.5l浓缩胶 母液0.3ml+凝胶缓冲液0.93ml+双蒸水1.27ml+10%AP25l+TEMED2.5l分离范围 1100KD，最佳分离范围 270KD初始电压30，等