Western blots on worm lysates

SternM Lab SternM.Lab at quickmail.yale.edu
Mon Apr 20 20:55:39 EST 1998


                       Subject:                               Time:4:35 PM
  OFFICE MEMO          Western blots on worm lysates          Date:4/20/98

Hi everyone!

I have an interesting problem to toss out to the community.  I am hoping
someone else had a similar problem (and solved it!)

In our lab we have made antibodies to two transmembrane proteins.  In both
cases multiple antibodies were made for each protein using different sites
in the protein to make bacterial fusions.

Both cases we made polyclonal rabbit antibodies.

Here is the problem.  In one case the antibodies were not purified but
worked well on the bacterial fusions and on the worm protein when it was
expressed in tissue culture cells.  This antisera was good for westerns and
for IPs on the protein in tissue culture cells.  However, it will not
recognize the protein in a worm lysate western.

The antisera against the second protein was affinity purified and both non
purified and purified sera work extremely well against bacterial fusions.
Again, I cannot recognize the protein on western blots using worm lysates.
In this case I have three separate antibodies to try, and none of them work.

I have used antibodies to a cytoplasmic worm protein and can show that my
lysates are fine, at least as far as Goa-1 is concerned. 8)

I realize that this may be a protein expression problem, in that these two
proteins are may not be expressed at high enough levels to do a straight
lysate.

Has anyone else had similar problems?

I am currently trying:
1) To do membrane preps as in the pat-3 paper
2) To use a GFP tagged version of the transmembrane protein and probing the
blots with anti-GFP antibodies as a control
3) To express the second protein in tissue culture cells in order to see if
the antibodies can detect it on a Western blot or IP the protein.
4) I haven't tried whole worm staining, partially because I'd like to be
comfortable about detecting the protein with the abs before I go to this,
but I am open to the possibility if people think this is a good idea.


Thanks for sticking with this long convoluted message!  If anyone has any
insights, similar experiences, or potential helpful advice, I would love to
hear them!

Thanks again,
Rebecca
(Stern Lab)





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