Antibody recognition of a protease created epitope

Emir Khatipov khatipovNO at NOuchicago.edu
Sun Dec 2 22:49:27 EST 2001


Artem,
Could you enlighten me on those biotin accepting peptides? They might be
very useful for me. What are they and in what host they are getting
biotinylated (in vivo?) Did someone use them for attaching biotin to
non-biotin-dependent proteins just by introducing a specific motif (I did
some searching in PubMed but did not find anything relevant so far)? --- If
I did not get you wrong of course.
Emir Khatipov



----------------------------------------------------
"Artem Evdokimov" <AEVDOKIMOZ at cinci.rr.com> wrote in message
news:uWVN7.83197$z55.10181580 at typhoon.neo.rr.com...
One of the FLAG antibodies will only recognize FLAG when it's not within the
context of an intact protein chain. This will require mutaitons or
insertions in your sequence plus the cut will have to be made exactly at the
beginning of the FLAG sequence.

If you wish to go crazy, you could try in-vitro transcription with altered
(fluorescent) amino acid :)

If you wish to be a bit less nuts, you may want to try biotinylation
(biotin-acceptor peptides will get auto-biotinylated on a specific lysine)
but that would mean that you'd insert a 10+ residue peptide into your
sequence, not necessarily good.

You can label lysines with stuff but if you do it in vivo, then all the
lysines will be uniformly labeled, not very nice I suspect.

Could you elaborate a bit more on your problem ?

A.G.E.
""Gregory O'Sullivan"" <osullivan at mpih-frankfurt.mpg.de> wrote in message
news:002e01c175e6$9473a7e0$0e05058d at gregory...
Hi,

I am trying to identify a protein after cleavage by a protease and follow it
as oppsed to the intact version.

One way would be to use a commercially available antibody that will
recognise a defined set of residues only after proteolytic cleavage and not
in the uncleaved protein. Thus, cleavage of the peptide bond will leave a
new N-terminus followed by a series of residues which will create a new
epitope not present in the intact protein. Does anybody know of such an
antibody ?

Alternatively, perhaps is there an 'in vivo', non-toxic way of labelling an
amino acid (e.g. lysine) with a free N-terminus ?

Any suggestions will be greatly appreciated.


Gregory A. O'Sullivan PhD.
Dept. of Neurochemistry,
Max Planck Institut fur Hirnfornschung,
Deutschordenstrasse 46,
D-60528 Frankfurt/Main,
Germany
Tel: +49 69 96769222
Email: osullivan at mpih-frankfurt.mpg.de





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