I have no reference on me right now (I am at home) however the gist of it is
that you use 11-13 residue sequence which gets biotinylated on a specific
lysine. There are a couple of articles in medline, dealing with the sequence
requirements. Biotinylaiton is quite sequence-specific, however it is not
complete unless you overexpress the relevant biotinyl-transferase. This is
probably the reason why the technique did not get too popular.
Theoretically, it can result in 95%+ purity after one step (monomeric avidin
column). Practically, monomeric avidin columns are a tad expensive, so it's
best to make them instead of bying them - this is possibly another reason
why the technique never got too popular.
If this isn't enough for you to locate the specifics, write me personally
and I will dig up some more data, or relay you to someone who knows more :)
Artem
"Emir Khatipov" <khatipovNO at NOuchicago.edu> wrote in message
news:2CCO7.34$t4.842 at news.uchicago.edu...
> Artem,
> Could you enlighten me on those biotin accepting peptides? They might be
> very useful for me. What are they and in what host they are getting
> biotinylated (in vivo?) Did someone use them for attaching biotin to
> non-biotin-dependent proteins just by introducing a specific motif (I did
> some searching in PubMed but did not find anything relevant so far)? ---
If
> I did not get you wrong of course.
> Emir Khatipov
>>>> ----------------------------------------------------
> "Artem Evdokimov" <AEVDOKIMOZ at cinci.rr.com> wrote in message
> news:uWVN7.83197$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>>
