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 2×Tricine sample buffer for

2×SDS 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 manufacturer’s 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, Tris⋅Cl/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 solution b  Separating Gel Staking gel

30% acrylamide/0.8% bisacrylamide 9.80 ㎖  1.62 ㎖ 

Tris⋅Cl/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 ㎕  5 ㎕ 

aThe 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)

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

cBest 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℃ 

2×Tricine sample buffer

2 ㎖  4×Tris⋅Cl/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 ㎖ 

4×Tris Cl/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℃ 

Tris Cl/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℃