小中大
tris-glycine gels 15%的胶最佳分离范围为6.5~200KD
所以你的3KD的蛋白很难分离开来。
建议使用Tris-tricine gels，
16.5% 分离范围 1～100KD
最佳分离范围 2～70KD
以下的PROTOCOL是最为流行的Tris-tricine 系统（Separating gel中使用的是甘油，少数人加入尿素），我使用过的,效果很好！（见下文， 分为两个部分：1. Stock Solutions for Tris-Tricine-SDS-PAGE (16.5% T, 6.0% C)；2. Immediately Used Solutions for Tris-Tricine-SDS-PAGE (16.5% T, 6.0% C)）( 各个组分的量可以成比例变化)（格式有些变化，请整理一下）（乱码的那个字为微micro ）
当您有疑问时可参考原始文献（目前几乎所有的Tris-Tricine-SDS-PAGE 都是参考了以下网络硬盘上的这篇文献），原始参考文献为两位的德国人发表在Anal. Biochem.上题为Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. 。到这里下载：cuturl('http://free.ys168.com/?ajzhangyu')。 dxy 求助文件夹中，题为Tris-Tricine SDS-PAGE.pdf
Stock Solutions for Tris-Tricine-SDS-PAGE (16.5% T, 6.0% C)
Anode buffer (1 h)
MW 500 ml
0.2 M Tris, pH 8.9 121.14 12.11 g
Dissolve 12.11 g Tris base in 200 ml dH2O. Adjust pH to 8.9 with 1 N HCl. Add dH2O to 500 ml total volume. Filter the solution through a 0.45 µm filter and store at 4°C.
Cathode buffer (1-2 h)
MW 500 ml
0.1 M Tris, pH 8.25 121.14 6.055 g
0.1 M Tricine 179.2 8.96 g
0.1% (w/v) SDS 288.38 0.5 g
Dissolve 6.055 g Tris base and 8.96 g tricine in a total volume of 500 ml dH2O. Filter the solution through a 0.45 µm filter, add 0.5 g electrophoresis-grade SDS, and store at 4°C. It is not necessary to adjust the pH of this buffer, which should be around 8.25.
Gel buffer (10 h)
MW 500 ml
3.0 M Tris, pH 8.45 121.14 181.65 g
0.3% SDS 288.38 1.5 g
Dissolve 181.65 g Tris base and 1.5 g electrophoresis-grade SDS in 300 ml dH2O. Adjust the pH of the solution to 8.45 with concentrated HCl. Bring to final volume of 500 ml with dH2O. Store at 4°C.
Separating gel monomer (49.5% T, 6.0% C) (2-3 h)
MW 200 ml 300 ml
46.5% (w/v) Acr 71.08 93.0 g 139.5 g
3.0% (w/v) Bis 154.17 6.0 g 9.0
Mix 93.0 g acrylamide and 6.0 g N, N'-methylene-bisacrylamide in a total volume of 200 ml dH2O. Filter solution through a 0.45 µm filter and store at 4°C protected from light. Discard after 30 days.
CAUTION: Acrylamide and bisacrylamide are potent neurotoxins and are absorbed through the skin. Wear a mask while weighing the powder. Gloves and a lab coat should be worn when handling the solution. Do not mouth pipette.
Stacking gel monomer (49.5% T, 3.0% C) (2-3 h)
MW 100 ml 200 ml
48.0% (w/v) Acr 71.08 48.0 g 96.0 g
1.5% (w/v) Bis 154.17 1.5 g 3.0 g
Mix 96.0 g acrylamide and 3.0 g N, N'-methylene-bisacrylamide in a total volume of 200 ml dH2O. Filter solution through a 0.45 µm filter and store at 4°C protected from light. Discard after 30 days.
Immediately Used Solutions for Tris-Tricine-SDS-PAGE (16.5% T, 6.0% C)
Separating gel (16.5% T, 6.0% C)
One-pack gel(3.3 ml)
Separating gel monomer 1.10 ml
Gel buffer 1.10 ml
glycerol 0.34 ml
ddH2O 0.76 ml
10% (w/v) APS 33 l
