Summary: E-63 WESTERN BLOT
(Protein techniques/ Western blot/ Last update 3/15/97/ Martina)
1. Clean the gel plates with EtOH.
2. Pour the separating gel. Leave about 1 cm space below the comb for stacking gel. You'll
need about 4 ml of separating gel/ minigel.
Overlay carefully with H2O, and let polymerize for about 20 minutes.
3. Pour off the H2O, insert the comb, and pour the stacking gel. Less then 1 ml
Let polymerize for about 20 minutes before runing the gel. Alternatively, wrap in the wet paper
towel and Saran Wrap and store at 4°C (after 2 days it is still OK, haven't tried longer storage).
4. If you are running two gels at the same time, start at 36 mA. After samples enter the
separating gel, increase to 46 mA or more, depending on how fast you want to be done (with
36mA --> 46 mA current, you will be done in about 1 hour).
Recipe for 10 ml of 12% gel
H2O 3.3 ml
30% acrylamide mix 4.0 ml
1.5 M Tris, pH 8.8 2.5 ml
10% SDS 0.1 ml
10% APS 0.1 ml