Western Blot Procedure
1. Run ~ 50 _g of whole cell extract on a PAGE gel (ie: 4-12% NuPAGE Bis-Tris gel).
2. Transfer the protein from the gel to a nitrocellulose membrane for 45-60 minutes.
3. Block the membrane with blocking buffer [1X TBS / 0.1% Tween-20 / 5% NFDM] for 1 hour at room temperature.
4. Rinse the membrane with 1X TBS, twice.
5. Dilute the 1_ antibody (cat# NB100-168, anti-MGMT) 1:1,000 to 1:3,000, in dilution buffer [1X TBS + 1% BSA].
6. Incubate the membrane with 1_ antibody for 1 hour at room temperature.
7. Wash the membrane with washing buffer [1X TBS + 0.05% Tween-20] 1 X 15 minutes, 3 x 5 minutes.
8. Dilute 2_ antibody [anti-mouse-HRP] in dilution buffer, add to the membrane, and incubate for 35 minutes at room temperature.
9. Wash the membrane with washing buffer 1 X 15 minutes, 3 x 5 minutes.
10. Develop the membrane with an ECL kit (ie: ChemiGlow Chemiluminescence reagent from AlphaInnotech].
