In a 25 ml side-arm flask, mix acrylamide solution, Tris-Cl/SDS, and ddH2O. Add glycerol to separating gel only. Very Important especially for the stacking gel !!--> Degas under vacuum and sonication for 10 - 15 minutes. Add 10% ammonium persulfate and TEMED. Swirl gently to mix, use immediately. After pouring the separating gel, quickly add ~100 ul of water saturated isobutyl alcohol to each gel. Let gels polymerize for at least one hour undisturbed. Then prepare and pour the stacking gel.

Buffers

Tris-Cl/SDS (3M Tris-Cl, 0.3% SDS, pH8.45)

Dissolve 182 g Tris base in 300ml ddH2O. Adjust to pH8.45 with HCl. Add H2O to 500ml total volume. Add 1.5 g SDS and store at 4 C.

Gel Running Reagents

1x Cathode Buffer (Load on top into wells)

12.11 g Tris base
17.92 g tricine
1 g SDS
Dilute to 1 liter with ddH2O
Do not adjust pH
Store at 4 C
Final concentrations are 0.1M Tris, 0.1M Tricine, and 0.1% SDS

5x Anode Buffer (Load bottom w/ 1x, gel apparatus tray)

121.1 g Tris base
500 ml H2O
Adjust to pH 8.9 with concentrated HCl
Dilute to 1 liter with ddH2O
Store at 4 C
Final concentration is 0.2M Tris-Cl, pH8.9

Coomassie blue staining solution

100 ml acetic acid
900 ml ddH2O:Methanol (1:1)
2.5 g Coomassie blue G-250
Store at room temp

Destain Solution

100 ml acetic acid
900 ml ddH2O:Methanol (1:1)

2x Sample buffer

1 ml 1M Tris-Cl pH 6.8
0.4 g SDS
2 ml glycerol
0.02 g bromophenol blue
0.31 g DTT (depending on protein cysteine content; not needed for protein L and SH3) Adjust volume to 10 ml

2x Tricine sample buffer

1 ml 1M Tris-Cl ph 6.8
2.4 ml (3g) glycerol
0.8 g SDS
2 mg Coomassie blue G-250
0.31 g DTT (depending on protein (cysteines); not needed for protein L and SH3) Adjust volume to 10 ml

Samples (~5-10ul of protein prep) should be mixed with sample buffer and boiled for 5 minutes before loading on gel. Run gels at a constant voltage of 100-150 V until the dye front reaches the bottom of the gel. To stain gels, add gel to staining tray w/ enough stain solution to cover gel. You may stain overnight at room temp or you may quick stain by microwaving stain/gel for ~10 seconds until solution is sufficiently warm (do not boil), then let stand for ~10 minutes. To destain remove gel from stain solution, add to new tray, and add destain solution until gel is covered. Kimwipes may be added to absorb the stain. Destain overnight at room temp w/gentle shaking or you may quick destain by microwaving destain/gel for 10 sec until sufficiently warm (do not boil), and let stand w/gentle shaking (heating can be repeated). Staining and destaining should be done under the hood.