2024年4月13日发(作者：)

Tricine-SDS-PAGE具体步骤

有篇Nature的protocal，我们试验室的Tricine-SDS-PAGE就是

参照其做的。

Tricine sds (209.29k)

Tricine-sds-page

一.试剂及缓冲液

AB-3 浓缩胶: 9.6g丙烯酰胺0.3g甲叉双丙烯酰胺溶于20ml

水.(49.5%T,3%Cmixture)

AB-6 分离胶: 9.3g丙烯酰胺0.6g甲叉双丙烯酰胺溶于20ml水.

(49.5%T,6%Cmixture)

凝胶缓冲液: 1X 2.422gTris 0.02gSDS溶于20ml水,HCL调节

pH=8.45.

样品缓冲液:1X

12%SDS,6%mercaptoethanol,30%glycerol,0.05%coomassie blue

G-250,150mMTrisHCL(pH=7.0)

阴极缓冲液: 1X 1.0MTris 1.0MTricine 1%SDS pH约8.25,基本不

用调.

阳极缓冲液: 1X 1.0MTris 0.225MHCL pH=8.9, HCL调节.

固定液: 250ml甲醇,50ml冰醋酸, 0.05mol醋酸铵加水定容

500ml.( 0.7mm,30min)

染色液: 0.025%考马斯亮蓝G-250 10%冰醋酸. ( 0.7mm,60min)

脱色液: 10%冰醋酸. ( 0.7mm,2h或者过夜)

二.配胶: 0.07×14×14cm 两块胶的用量.

(一) Separating gel 16%gel

1. AB-6: 5ml

2.凝胶缓冲液: 5ml

ol: 1.5g

4.上述加水至15ml

5.10%APS 50ul

5ul

(二)Spacer gel 10%gel

1. AB-3: 600ul

2.凝胶缓冲液:1ml

ol: 0.3g

4.上述加水至3ml

5.10%APS 15ul

1.5ul

(三)Stacking gel 4%gel

1. AB-3: 500 ul

2.凝胶缓冲液: 1.5ml

3.上述加水至6ml

4.10%APS 45ul

4.5ul

三.注意:

1.电压:进入分离胶之前30V,之后100 V,在跑至分离胶底部之前可

以调至200V.注意温度不能超过35~40℃.

2.本实验适合小蛋白、多肽的分离.1~100kD.

3. Spacer gel 10%gel的用途是为了使1~5 kD的小分子得到更

好的分离.是否配制,按照自己需要.

4. 这是我做了半年摸索出的条件.效果非常好.用它已经很好地分离

出我的目标宝贝.和大家一起分享.

以下是详细说明书：

Tricine/Polyacrylamide Gel Electrophoresis

Used for pilin processing analysis but generally useful for

resolution of small (15-35 Kdal) proteins of similar size.

See

Strom, M. S., D. N. Nunn, and S. Lory. 1993. A single

bifunctional enzyme, PilD, catalyzes cleavage and N-methylation

of proteins belonging to the type IV pilin family. Proc. Natl. Acad.

Sci., 90:2404-2408.

or

Strom, M. S., and S. Lory. 1992. Kinetics and sequence

specificity of processing of prepilin by PilD, the Type IV leader

peptidase of Pseudomonas aeruginosa. J. Bacteriol. 174:7345-

7351.

Original citation: Schagger, H. and G. von Jagow. 1987.

Tricine-sodium dodecyl sulfate-polyacrylamide gel

electrophoresis for the separation of proteins in the range from

1 to 100 kDa. Anal. Biochem. 166: 368-379.

Reagents:

Anode Buffer ( ): 200 mM Tris pH 8.9 (can dilute from 10X

stock)

Cathode Buffer (-): 100 mM Tris/100 mM Tricine/0.1% SDS

(no need to pH, but will be ~8.25) these can be made as 10X

stocks and diluted before use

Gel Buffer: 3.0 M Tris, pH 8.45/0.3% SDS

Note: the pH's given for the anode and gel buffers are

essential

Stacking acrylamide: 48% acrylamide/1.5% bis-acrylamide

Separating

acrylamide

Sample buffer: (add equal volume to sample), for 20 ml:

5 ml 0.5 M Tris, pH 6.8

4 ml 20% SDS

1 ml 2-mercaptoethanol

4 ml 50% glycerol

0.004 g bromophenol blue

6 ml water

For each minigel (Hoeffer "Mighty Small" or BioRad "Mini

acrylamide: 46.5% acrylamide/1.5% bis-

Protean-II"--scale up as required):

1. Separating gel:

15% gel

2 ml separating acrylamide

2 ml gel buffer

2 ml 50% glycerol

10% gel

1.22 ml separating acrylamide

2 ml gel buffer

2 ml 50% glycerol

0.78 ml water

polymerize with 75 ul 10% APS and 7.5 ul TEMED

2. Stacking gel:

0.25 ml stacking acrylamide

0.75 ml gel buffer

2.0 ml water

20 ul 10% APS

2 ul TEMED

Polymerize both the stacking and separating gels at the

same time (I used small disposable tubes), and pour stacking gel

directly onto the separating gel (pour carefully but quickly--they

won't mix but the separating gel polymerizes within 2-3 minutes).

Make each separating gel mixture separately and add TEMED and

APS right before pouring. Multiple stacking gel mixtures can be

made in the same tube, but you have around 10 minutes before

these start to polymerize too.

Sample loading and electrophoresis:

For the minigels, 5-10 ul per well gives best results. Layer the

samples in the well very carefully, and be sure to flush out any

unpolymerized acrylamide before loading.

Electrophorese at 25-35 mA (constant current) per gel in

minigel setup. Foam will appear on the top (cathode), and

additional cathode buffer may have to be added during the run.

好东西

我的Tricine-SDS-PAGE电泳图，在大分子处有条带，在小分子处

无条带，是什么原因

1、可能分离胶浓度太小，把胶的浓度加大，跑的时候电压小点

2、小分子蛋白含量少，可以加大蛋白上样量

谢谢luhao82 ，我再按你的建议试试，但是我都是按你的步骤做

的啊