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Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. Prepare solutions with reverse osmosis deionized RODI or equivalently purified water. Treat cells by adding fresh media containing regulator for desired time. Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube.

Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature.