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

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Primary Antibody Etudianr Buffer: 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. The membrane is now ready to reuse. Wash three times for 5 min each with 15 ml of TBST.