Polyclonal screening bkg.

Michael Benedik bchs1b at Elroy.UH.EDU
Wed Jan 27 00:02:55 EST 1993


In article <1993Jan25.234505.24318 at midway.uchicago.edu>, jm68 at quads.uchicago.edu (James R. Mensch Jr.) writes:
>We are having a problem with high background in our attempts to screen a
>lambda ZAPII cDNA library.  The instructions accompanying the library, the
>protocol obtained with the screening/detection kit from the same source as the
>library as well as that from another vendor, and the technical services people
>have all been consulted to no avail.  Though we are not rookies at this game,
>perhaps there is some good trick we haven't heard of yet.  The parameters of
>our system are these:
>- A lambda ZAP-II rat liver cDNA library is plated on XL1-Blue bacteria
>- IPTG-saturated (10 mM) nitrocellulose filters, S&S BA85,are used to prepare
>  plaque lifts (2 sets).
>- Rinsing/washing of filters is done in 'TNT'(10 mM Tris/150mM NaCl/
>  0.05% Tween 20, pH 8.0); blocking in TNT with 6% BSA added.
>- Primary antibody is a rabbit polyclonal. The preparation in use was 'stripped'  of species reactive to E.coli/phage proteins by incubation with filters
>  bearing proteins from a bacterial/phage lysate.
>- Secondary antibody is goat anti-rabbit with conjugated alkaline phosphatase,
>  signal production is by incubation with BCIP/NBT.
>Prior to use of the stripping lysate, an extremely heavy background was obtainedon all primary round filters.  Post-stripping this background was reduced to a
>light violet tinting with non-positive (ie. all) plaque images discernable.
>Over time this preparation has yielded progressively heavier background to the
>point where the plaque images are now seen as lighter spots!  Negative negativesif you will.  These effects are independent of reagent batches, solutions, etc.
>etc..  We are contemplating adding an intermediate antibody to the system.
>We have used a monoclonal to screen filters from the same filter lot lifted 
>from plates of the same batches as used with the polyclonal, the monoclonal gives no background problems albeit using a HRP-conjugated anti-mouse 2nd Ab.
>No definite positives yet either!  A positive control phage (globin cDNA insert,
>but detected by a monoclonal recognizing the B-gal portion of the fusion proteinproduct) gives a good signal against a field of negative (gt10) plaques.  Thus
>the polyclonal Ab prep appears to be the culprit, yet it works well with West-
>erns as regards binding to the target protein.
>Any ideas for us?  Thanks in advance for your consideration.  Jim M.
><j-mensch at midway.uchicago.edu>
>-- 
>===============================================================================
>Jim Mensch : j-mensch at uchicago.edu
>             menche at biovax.uchicago.edu
>===============================================================================

In reading your post I notice you say the background in getting worse with
time. I wonder if there is some degradation of the antibodies occurring.
Have you gotten a fresh aliquot of your serum from the freezer and trying
that?
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 Michael Benedik				INTERNET: Benedik at uh.edu
 Dept. of Biochemical & Biophysical Sciences	
 University of Houston				BITNET: Benedik at uhou
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