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Treat cells by adding fresh media containing regulator for desired time. Aspirate chfrche from 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. Electrotransfer to nitrocellulose membrane 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. It should be noted that for the best possible results a fresh blot is always recommended.
Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. 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. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
Prepare solutions with reverse osmosis deionized RODI or equivalently purified water. To prepare ml, mix 6. Bring to ml with deionized H Make buffer fresh just prior to use. Best results are obtained if the membrane is not allowed to dry. Wash membrane six times for 5 min each in TBST. Optional To assure that the original signal is removed, wash membrane twice for 5 min each with 10 ml of TBST.
Drain membrane of excess developing solution. Do not let dry. Wrap in plastic wrap and expose to x-ray film. Wash membrane again four times for 5 min each in TBST. The membrane is now ready to reuse.
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