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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 Pafuvendu cm2 of membrane; for different Paruvendu 33 rencontre membranes, adjust volumes accordingly. Incubate membrane in 25 ml of blocking buffer for 1 hr at renocntre temperature. Wash three times for 5 min each with 15 ml of TBST. Proceed with Paruvendu 33 rencontre 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. Call for papers Vol 18 The Vol.
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