I thought I would post the results of my inquiry on anti-B-gal antibodies
which work well on fixed C. elegans.
Promega Mouse anti-B-gal Z3781
Reported by many people to work well on fixed C. elegans expressing
B-gal fusions. Try 1:100 dilution to start. Doesn't last well at
+4 degrees - freeze in aliquots. Also reported to work well on Westerns.
Sigma Mouse anti-B-gal monoclonal G-6282
Reported to work well on fixed animals at 1:300 dilution.
Cappel Rabbit polyclonal Purified anti-B-gal 55976
Cappel labs
Organon Teknika Corp.
919-620-2000
We use this on fixed animals in our lab at 1:100. Good if you need
a non-mouse source.
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Several months ago I requested help on various aspects of polyclonal
antibody development. I thought I would post what I found to be the
most helpful suggestions.
To have all the unpleasant work done for you by a group of proven
professionals, send purified protein to: East Acres Biologicals
Southbridge MA. Ask for Mindy Donovan 508-765-9580.
They will send you serum from innoculated animals after each boost.
Several people reccommended them, and I was very pleased with their
service.
Two techniques can work wonders on cleaning up anti-sera to remove
all contaminating Abs.
Positive selection for antibodies against your fusion protein:
Affinity purification. I found that for insoluble proteins,
6HIS Fusions purified under denaturing conditions can be used
right on the Ni-NTA column after washing away denaturant. Crude
anti-sera can than be bound, washed, and eluted on this same
column, eliminating the need to elute and dialyse fusions before
binding to a suitable matrix. Described in Biotechniques 17(2):257.
Negative selection: Acetone powders of E. coli and C. elegans (WT
or mutants) can remove unwanted Abs from anti-sera. Good for
primaries and secondaries. Described in the upcoming chapter by
David Miller in the C. elegans Methods in Enzymology book.
Together these two techniques made very messy anti-SEL-1 Abs
give very clean results.
Thanks for the help.
Barth
