12% - 14.2 KD trails slightly behind dye front.

1. Assemble the gel plates with spacers that match the thickness of the comb you plan to use. Clamp the glass sandwich with black clamps. Locate a comb of matching thickness. Seal the edges of the glass sandwich with molten agarose; this is easier than sealing with tubing and cleaner than grease.
2. Prepare the running gel. The recipes are for gels that are about 10cm X 10cm. Simple geometry will allow you to calculate how much solution must be made for larger gels. Be certain to use 0.4% SDS, 1.5 M Tris-Cl pH 8.8. All reagents should be of electrophoretic grade or high purity. Mix the following in a graduated cylinder.

Gel composition	10%	11%	12%
29.2% Acrylamide, 0.8% Bisacrylamide	6.67 ml	7.33 ml	8 ml
0.4% SDS, 1.5M Tris-Cl pH8.8	5 ml	5 ml	5 ml
distilled water	8.33 ml	7.66 ml	7 ml

3. Add 67 ul of 10% ammonium persulfate (10% stocks are stored at -20^oC) and gently swirl the solution.
4. Add 10 ul of TEMED (stored in refrigerator), cover with parafilm, and gently invert several times to thoroughly gel components (avoid introducing air bubbles as this can inhibit polymerization).
5. Immediately pour the acrylamide solution between the plates. Fill the space up so there will be enough room to form a stacking gel of 0.5 to 1 cm.
6. Overlay the acrylamide with water-saturated isobutanol to a depth of a few millimeters.
7. Allow the gel to polymerize for 1 hour. Don't leave it overnight. A discontinuity in the gel forms that distorts the mobility of the proteins.
8. While the running gel is polymerizing, prepare the running buffer for the gel tanks. Combine in a total volume of 1 liter: 3.03 g Tris base, 1 g Ultrapure SDS, 14.4 g glycine and water.
9. Prepare the protein samples for electrophoresis. Take an adequate amount of 2.5 X SDS-PAGE sample buffer (250 mM Tris-Cl pH 6.8, 5% SDS, 0.25% bromophenol blue, 25% glycerol) and add 1/5 volume of 1M DTT. This gives 2X with reducing agent. If the protein is already in solution, add an equal volume of 2X sample buffer. If the protein has been precipitated with TCA, residual acid often remains even after an acetone wash. These precipitates should be dissolved in 50 mM Tris-Cl (pH 6.8), 100 mM Dithithreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol, 25 mM NaOH.
10. After the running gel has polymerized, rinse the isobutanol from the surface with copious amounts of water. Drain excess water.
11. Prepare the stacking gel. This is composed of 5% acrylamide (I used to use 4.75% but this often fails to polymerize completely). Be certain to use 0.4% SDS, 0.5 M Tris-Cl pH 6.8. Mix the following:
• 1.67 ml of 29.2% Acrylamide, 0.8% Bisacrylamide
• 2.5 ml of 0.4% SDS, 0.5M Tris-Cl pH 6.8
• 5.83 ml of distilled water 5.92 ml
• Add 50 ul of 10% ammonium persulfate, mix
• add 10 ul of TEMED, mix
• Pour between plates and carefully insert clean comb so as to avoid air bubbles.
12. Polymerize stacking gel for 30 to 60' before removing comb and running gel.
13. Carefully remove comb while lubricating the top area with running buffer (see below)
14. Rinse wells thoroughly with running buffer and assemble the gel in the electrophoresis rig.
15. Boil protein samples for 5 minutes and then load the gel.
16. Run gels at 150 to 200 volts.

1. Immerse gel for 1 hr in 51.4 ml EtOH/120 ml H₂O/28.5 ml 35% Formaldehyde/228 mg Coomassie.
2. Destain gel with 200 ml portions of a mixture containing 250ml EtOH/750ml H₂O/10ml Formaldehyde. Change the destain solution when it becomes very blue.
3. Wash gel with several changes of H₂O.
4. Soak gel in 100 ml of 10% acetic acid, 1% glycerol for 20 minutes
5. Dry for 1 to 2 hours with gel drier set at 80 ^oC and cycle set on the trapezoid shape