Western Blot Protocol for MGMT Antibody (NB100-168)

Procedure Guide for NB 100-168 Monoclonal anti-MGMT

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].