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Dot blot on bacteria


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#1 mel4-7

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Posted 09 April 2004 - 07:51 AM

Hi,
I have to screen many clones of many expressed proteins in BL21 DE3 after
induction with IPTG.

Does someone already try to make a dot blot for screening of expressing clones? I think that I we would be more easier and simple instead of making many SDS PAGE. But I'm not sure about the method to lyse bacteria. Do you think SSB will be sufficient before droping in nitrocellulose? (and probe with a anti-his tag)

Thanks
Mélanie

#2 phdconsult

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Posted 10 April 2004 - 06:14 AM

Mel

You can do a colony dot blot but solid state expression is never definitive- you might miss a perfectly good clone. We suggest doing at least 3 screens with cells induced at 3 different temperatures (25/37/42) and then pick clones that show up more than one time. A HisTag screen combined with a screen that uses a monoclonal antibody to target protein might be pertinent-that way non-specific signals are eliminated. Also, do you have the IPTG-Xgal option available to you?

To answer your question, SSB is sufficient to lyse cells.

phdconsult




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