Immunoblot Protocol

1. Perform SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a cell nuclear extract (Extraction buffer: 20mM HEPES, pH 7.9; 25% glycerol; 0.42 M NaCl; 1.5 mM MgCl₂; 0.2 mM EDTA; 0.5 mM PMSF; 0.5 mM DTT) and transfer the proteins to nitrocellulose. Wash the bottled nitrocellulose twice with water.
2. Block the blotted nitrocellulose in freshly prepared PBS containing 3% nonfat dairy milk (PBS-MILK) for 1 hour at 20-25 ̊C with constant agitation.
3. Incubate the nitrocellulose in 0.5-2μg/mL of anti-Myc Tag, clone 9E10 diluted in freshly prepared PBS-MILK overnight with agitation at 4 ̊C.
4. Wash the nitrocellulose twice with water.
5. Incubate the nitrocellulose in the appropriate secondary reagent (a goat antimouse HRP conjugated IgG --Cat # 12-349, 1:2000 dilution) in PBS-MILK for 1.5 hours at room temperature with agitation.
6. Wash the nitrocellulose twice with water.
7. Wash the nitrocellulose in PBS- 0.05% Tween 20 for 3-5 minutes
8. Rinse the nitrocellulose in 4-5 changes of water.
9. Use detection method of choice (enhanced chemiluminescence is preferred)