Dry membrane to do western blotting - (Jun/28/2005 )

Hi all,

I am trying to do westernblotting by using the membrane that was alreday used for an antibody and developped. Do I need to boil the membrane or just start from blocking. Thanks

Steer

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hi
i would advise you to strip your membrane and again block follow by analysis.
Two discussions may help :

http://www.protocol-online.org/forums/inde...897&#entry11897
http://www.protocol-online.org/forums/inde...182&#entry18182

fred

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What type of membrane is it? If its PVDF you need to wet with Methanol first and then you can rehydrate in water followed by PBST. If nitrocellulose, you can rehydrate directly in PBST.

Cheers,

LTR

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The first step is to rehydrate membranes if they are dry. If you're working with PVDF membranes, do it in 40% methanol for 30 minutes with shaking (or T-PBS or T-TBS for nitrocellulose membranes). Then, you'll have to strip the membranes (to get rid of the previous Ab's) in a b-mercaptoethanol solution (the regular stripping protocol). After that, you are ready for blocking. Good luck.

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