|Before using the antibody to screen a library, it is important
to check its specificity on Western blots and to determine the optimum dilution factor and incubation conditions.
It is important to check that there is minimal crossreaction with
proteins in an E. coli lysate to avoid too high a background. If the antibody does show
some recognition of bacterial proteins this can usually be pre-absorbed
out, and mild crossreactivity will be progressively reduced if
the antibody dilution is kept and reused in subsequent screens.
|The library screen is essentially a Western blot, so it is important to do a series of these to determine the
optimum incubation conditions (usually 1 hour at room temperature)
and the appropriate dilution factor for the primary antibody.
Excess antibody will only give an unworkable background. It is
also important to assess whether the washing conditions described
will be sufficient to detect only the protein of interest in a
blot using total cell extract. If a number of other proteins are
also detected, then the antibody is probably unsuitable to use
for screening since too many unrelated clones will be purified.
However, more stringent washing may improve the situation, and
if so, these conditions should also be used in the screen. Sometimes
a purified protein used to raise an antibody contains a low-level
contaminant that proves to be very antigenic, giving a bright
signal on the Western in addition to the protein of interest.
In this situation, it is possible to proceed, but when picking
plaques after the first-round screen, it will be important to
select both bright and faint spots to ensure that the interesting
clones are not overlooked.
|Another issue is that if the protein of interest is thought to
be covalently modified (phosphate, carbohydrate), then it is wise
to check that the antisera will still recognize the native protein,
since obviously these modifications will not be reproduced in
the bacterially made protein during the screen.
|Polyclonal antisera are often reactive with bacterial and phage proteins. This can
be tested by dotting some phage-infected bacterial lysate on the
corner of a Western blot prior to blocking or running some on
a spare lane in the initial SDS-PAGE. If a strong signal is observed, it will be necessary to preabsorb
out the reacting antibodies. This can be done by incubating three
to four strips of nitrocellulose (5 x 10 cm) in bacterial lysate
and then blocking them for 1 hour at room temperature before rinsing
three times in TBST. Dilute a reasonable amount of the primary
antibody 1:5 in TBST, and incubate this with one of the filters
for 15 min at room temperature. Remove the filter, and discard
and replace with another. Repeat until all of the prepared filters
have been used. Store the final antibody solution, and recheck
on a Western for the appropriate dilution and also that the cross-reactivity
has been reduced sufficiently.
|Top Agarose is much easier to use than top agar, which frequently
sticks to the nitrocellulose and tears when the filters are removed.