immunoscreening cDNA libraries

Dennis J. Templeton djt2 at po.CWRU.Edu
Fri Jun 19 16:02:02 EST 1992


In a previous article, MPB at CBR.DWE.CSIRO.AU (Mark Bradley) says:

>Dear Netters: Can you help? We are having problems with background
>when we screen out cDNA library with ascites (MABS). We are using a
>lambda ZAP library.  Any help/advice regarding absorbing out
>cross reactive antibodies (ie Ecoli/phage) would be most
>appreciated.
>
>Mark Bradley
>CSIRO, Div. Wildlife & Ecology
>Canberra, Australia
>email: mpb at cbr.dwe.csiro.au
>

Mark;

the obvious things come to mind, like purifying your IgG and using it 
at maximal dilutions. It is possible that the problem results from your
detection system (i.e. your second antibody or the protein A or the
avidin/biotin) rather than with the primary antibody.  If so, switching
detection systems might help

If you have a genuine cross-reactivity to some bacterial or phage 
protein, you might be able to neutralize this using an irrelevant phage
lysate.  i.e. lyse some coli with WT lambda,(concentrate the cells before
lysing them with chloroform) and mix in some of this with your antibody
diluent.  Alternatively you could bind these proteins to some scraps of
nitrocellulose (or do mock phage-lifts of plates infected with wild type
lambda) and then pre-soak your antibody solution with  these mock filters.

hope this helps,

dennis



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