PAGE in Tris-Tricine Buffer System

1. Prepare and pour the separating and stacking gels using the recipes in Table.
2. Prepare the sample(same as laemmli system), but substitute 2Tricine sample buffer for
2SDS sample buffer, and heat 30 to 60 min at 40.
3. Carefully remove the Teflon comb without tearing the deges of the wells. Rinse and then fill
wells with cathode buffer
4. Fill the lower buffer chamber of the electrophoresis apparatus with anode buffer, assemble
the unit, and attach the upper buffer chamber per manufacturers instructions. Partially fill
upper chamber with cathode buffer so that the wells are covered.
5. Load samples and run gel, but run 1 hr at 30 V(constant voltage) followed by 4 to 5 hr at 150

V(Constant voltage). Use a heat exchanger to keep the electrophoresis chamber at room
temperature.
Table. Recipes fot Tricin Peptide Separating and Stacking Gelsa
In a 50 side-arm flask, mix 30% acrylamide/0.8% bisacrylamide soution, TrisCl/SDS, pH 8.45, and H2O.
Add glycerol to separating gel only. Degas under Vacuum 10 to 15 min. Add 10% ammonium persulfate and
TEMED. Swirl gently to mix, use immediately. Failure to form a firm gel usually indicates a problem with the
persulfate, TEMED, or both

Stock solutionb

Separating Gel

Staking gel

30% acrylamide/0.8% bisacrylamide

9.80

1.62

TrisCl/SDS, pH 8.45

10.00

3.10

H2O

7.03

7.78

Glycerol

4.0 g(3.17 )

10% (w/v) ammonium persulfate

50

25

TEMED

10

The recipes produce 30 of separating gel and 12.5 of stacking gel, which are adequate for two gels of dimensions 0.75 mm

14 14. The recipes are based on the Tris-Tricin buffer system of Schagger and von Jagow(1987)
b

All reagents and solutions used in the protocol must be prepared with Milli-Q-purified water or equivalent.

Best to prepare fresh.

Materials
30% acrylamide/0.8% bisacrylamide
prepare as for 29:1 solution (above), but adjust acrylamide to 30 g and bisacrylamide to 0.8 g in a
100 volume.
Cathode buffer
12.11 g Tris base (0.1 M final)
17.92 g tricine (0.1 M final)
1 g SDS [0.1% final]
Dilute to 1 liter with H2O
Do not adjust pH
Store up to 1 month at 4
Anode buffer
121.1 g Tris base (0.2 M final)
500 H2O
Adjust to H 8.9 with concentrated HCl
Dilute to 5 liters with H2O
Store up to 1month at 4
2Tricine sample buffer
2 4TrisCl/SDS, pH6.8
2.4 glycerol (24% final)
0.8 g SDS (8% final)
0.31 g dithiotheritol (0.2M final)
2 Coomassie blue G-250 (0.02% final)
Add H2O to 10
4TrisCl/SDS, pH 6.8
Dissolve 6.05 g Tris base (final 0.5 M) in 40 H2O. Adjust pH to 6.8 with 1 N HCl. Add H2O to
100 . Filter through at 0.45 filter. Add 0.4 g SDS (final 0.4 %). Store up to 1 month at 4
TrisCl/SDS, pH 8.45
Dissolve 182 g Tris base (final 3.0 M) in 300 H2O. Adjust pH to 8.45 with 1 N HCl. Add H2O
to 500 . Filter through at 0.45 filter. Add 1.5 g SDS (final 0.3 %). Store up to 1 month at
4