Screning E.coli transformants with an antibody?

Fergus Doherty Fergus.Doherty at nottingham.ac.uk
Fri Feb 5 08:13:32 EST 1999


Hello, I hope you can help with this question.

I need to screen E.coli transformants rapidly and simpel for expresion of a
recombinant protein.  The Vector used provides ampicillin resistance but
there is no blue/white selection.  It is possible that I can get
transformants that don't contain my insert (but contain plasmid without
insert), so I need to screen a reasonable number of transformants with an
antibody to the recombinant protein.  I understand I could pick colonies
and transfer them to a gridded agar plate, and to a gridded nitrocellulose
filter, on top of an agar plate with IPTG to induce expression of my
protein.  Then the problem is lysing the cells in such a way as to fix the
proteins on to the filter prior to probing with antibody.

Can anyone sugest a protocol or tell me more about this method of screening?

Thanks

-- 
Fergus Doherty,
Dept Biochemistry,
Nottingham University,

Fergus.Doherty at nottingham.ac.uk
0115 970 9366 (74-41366 internal)



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