IUBio

anti-GST antibodies for fusion protein detection

C-Y Khoo cyk10 at cus.cam.ac.uk
Fri Nov 13 07:13:49 EST 1992


I too, have been looking for anti-GST antibodies, but without any success.
I called Pharmacia and they said that they are in the process of
developing
such a reagent, but that it won't be ready for some time yet. There are
commercial sources of anti-GST antibodies, but they're raised against the
human antigen, and according to Pharmacia, don't cross react with the S.
japonicum antigen. Your best bet is to express pGEX, purify GST from
the supernatant, and immunize a rabbit with it.

Depending on various things, I _may_ have some anti-GST by January next
year
(I'm currently immunizing with a fusion protein). Ask me later!

Regarding your expression problems, I found out that _most_ fusion
proteins
are insoluble and are found in inclusion bodies, _not_ the supernatant (as
stated in Smith and Johnson (or indeed, Current Protocols in Molecular
Biology). See Fiona Marston's articles in Biochem J. (1986) 240, 1-12 and
an article of the same name in another journal (don't have the reference
at hand, unfortunately!). Also, Nagai and Thogersen (1987) Methods Enzymol
153, 461-481 for inclusion body prep. As a first step, you may want to
boil some of the pellet to see if the fusion protein is actually being
produced, but in an insoluble form.

Good luck!

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Chong-Yee Khoo                             E-mail: cyk10 at cus.cam.ac.uk
Wellcome/CRC Institute                     Talk:  
cyk10 at cy-mac.welc.cam.ac.uk
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