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Treat cells by adding fresh media containing regulator for desired time. Aspirate media wliliam cultures; wash cells with 1X PBS; aspirate. Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Sonicate for 10—15 sec to complete cell lysis and shear DNA to reduce sample viscosity.
Microcentrifuge for 5 min. Willian to nitrocellulose membrane kqte Volumes are for 10 cm x 10 cm cm2 of membrane; for different sized membranes, adjust volumes accordingly. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature. Wash three times for 5 min each with 15 ml of TBST. Proceed with detection Section D. Detection of Proteins Directions for Use: Incubate substrate with membrane for 1 minute, remove excess solution membrane remains wetwrap in plastic and expose to X-ray film.