Tricine is a zwitterionic buffer for biochemistry and molecular biology, with a pH buffer range of 7.4-8.8. During SDS-PAGE to seperate small peptides, tricine is commonly used instead of glycine in the Laemmli buffer. Tricine-SDS-PAGE gel has superior performance for the identification of proteins with molecular weight below 10 kd. While conventional SDS-PAGE is used to separate these proteins, some problems may occur, such as vague strip, overly soft gel. Here we describe a preparation method for Tricine-SDS-PAGE gel.

Reagents:

Anode Buffer (+): 200 mM Tris pH=8.9

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

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

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

Separating acrylamide: 46.5% acrylamide/1.5% bis-acrylamide

Sample buffer (20 ml):

5 ml 0.5M Tris pH=6.8; 4 ml 20% SDS; 1 ml 2-mercaptoethanol; 4 ml 50% glycerol; 0.004 g bromophenol blue; 6 ml ddH2O.

Separating gel:

15% gel: 2 ml separating acrylamide, 2 ml gel buffer, 2 ml 50% glycerol.

10% gel: 1.22 ml sperating acrylamide, 2 ml gel buffer, 2 ml 50% glycerol, and 0.78 ml ddH2O.

The polymerization was carried out with 75 ul of 10% ammonium persulfate (APS) and 7.5 ul of tetraethylethylenediamine (TEMED).

Stacking gel:

0.25 ml of stacking acrylamide, 0.75 ml gel buffer, 2 ml ddH2O, 20 ul 10% APS, 2 ul TEMED.

The stacking and the separating gel should be polymerized at the same time, and then pour the stacking gel onto the separating glue (pour carefully but quickly).

Sample loading and electrophoresis:

For the minigel, 5-10 ul per well gives the best results.  Layer the samples in the well very carefully, and be sure to flush out any unpolymerized acrylamide before loading. After loading the sample, a constant current of 25-35 mA was set in the minigel setup for electrophoretic analysis. During the run, foam will appear on the top of the cathode and additional cathode buffer have to be added.

The above description of the preparation of Tricine-SDS-PAGE gel provides an example for researchers. But in a specific experiment, it is also necessary to find a suitable preparation method suitable for the experiment according to the actual situation.

Edited by Suzhou Yacoo Science Co., Ltd