Antibody recognition of a protease created epitope

Artem Evdokimov AEVDOKIMOZ at cinci.rr.com
Fri Nov 30 19:58:02 EST 2001


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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