Polyclonal screening bkg.
James R. Mensch Jr.
jm68 at quads.uchicago.edu
Mon Jan 25 18:45:05 EST 1993
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
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