Tris-Tricine gels (Small proteins / peptides) – Elizabeth Rico-Bautista, April 2006

Buffers:

1. Cathode buffer 1X (upper chamber): 100 mM tris, 100 mM tricine, 0.1% SDS

Tris-base: 6.055 g

Tricine: 8.96 g

SDS: 0.5 g

Final vol: 500 ml

2. Anode buffer 10 X (lower chamber): 200 mM tris pH 8.9

Tris-base: 121.1 g

pH 8.9 with HCl

Final vol: 500 ml

3. Tris-HCl/SDS buffer: 3.0 M tris, 0.3% SDS

Tris-base: 36.4 g in 60 ml H₂O

pH 8.45 with HCl

H₂O up to 100ml

Filter the solution

Add 0.3 g SDS

Degas the solution for 10-15 min and store at 4°C for up to 1 month

4. Prepare fresh APS 10%

Separation gel Stacking gel

10% (2 gels) 15% (2 gels)

AA 4.9 ml 7.5 ml 972 μl

Tris-HCl/SDS 5 ml 5 ml 1.86 ml

H₂O 3.5 ml 0.9 ml 4.67 ml

Glycerol 1.58 ml 1.58 ml --------

APS (10%) 75 μl 75 μl 40 μl

Temed 10 μl 10 μl 4.5 μl

Final vol: 15 ml 15 ml 7.5 ml

Important: Use fresh APS. Degas the separation and stacking gel solutions before adding APS and Temed (10-15 min).

The separation gel should polimerize within 30 minutes. The stacking gel usually takes longer.