MBP versus GST...and the winner is...

Gregory A. Cox gac at aretha.jax.org
Thu Apr 6 10:08:44 EST 1995


In article <Daniel.L.Burgess-0304951502120001 at 174.52.med.umich.edu>,
Daniel.L.Burgess at um.cc.umich.edu (Dan Burgess) wrote:

> MBP versus GST...and the winner is...

description deleted
 
> My questions are these:  Has anyone tried both?  Alternatively, if 
> you've had some experience with either, were you satisfied?  I'm not
> looking for the perfect system, but I would like my first attempt to
> have at least a 'snowballs chance in Michigan' of working.
>  
> Thanks for your time,
>  
> Dan Burgess 
> U of Michigan
> Dept. of Human Genetics
> 
> -- 
> Positional cloning...it's not the kill, it's the thrill of the chase.

Hi Dan,

   Long time no see... I have only my experience with the MBP fusion to
share. I made anti-dystrophin fusion proteins with the NEB vector and had
no problems generating or purifying the proteins (except for one region
that was highly cystein-rich and insoluble). As for the MBP being more
immunogenic than your fusion partner, I can only say that the antibodies
that were generated to my soluble fusion protein were incredible. I had
specific titers that worked well on immunoblots at 1/100,000 dilutions for
alkaline phosphatase secondaries and at 1/1,000,000-5,000,000 when using
amplifivcation schemes such as the abc kits or chemiluminescent staining
(and no, these dilutions aren't typos). Just imagine what fraction of this
rabbits total circulating antibodies were anti-dystrophin. I bled the
rabbit out after the fourth bleed so I didn't keep it long enough to see
if the high titer antibodies to such a highly conserved protein would have
caused any pathology. Hope this helps.

Gregory A. Cox
The Jackson Laboratory
gac at aretha.jax.org

P.S. Nice .sig file. However, I don't know if you can say that your work
was a true positional cloning project when you had a transgene integration
to help you get the answer (is that considered cheating in positional
cloning :-) ).



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