Hi Yongfu,
An interesting approach is to add a c-term biotin tag as a bio-sensor.
If the c-term of the target protein is accessible the biotin tag will
get biotinylated by BirA (co-expression may be necessary depending on
the expression system). You can then easily detect the target protein
by WB or pull it down with streptavidin beads. I know Darren Hart is
using this approach at EMBL-Grenoble.
On a similar subject, OPPF is using Carboxypeptidase A to remove the c-
term 6xHis tag (LysHisHisHisHisHisHis) only to leave a c-term Lysine.
Have a look at:
http://nar.oxfordjournals.org/cgi/content/full/35/6/e45
Hope this helps.
Vangelis
--
Vangelis Christodoulou
Analist C
Anastassis (Tassos) Perrakis lab
The Netherlands Cancer Institute
Dept. of Molecular Carcinogenesis - H2
Plesmanlaan 121, 1066 CX, Amsterdam
The Netherlands
tel: +31 (0)20 512 1951
fax: +31 (0)20 512 1954
Website: xtal.nki.nl/Tassos_group/
On Feb 7, 2008, at 22:28, Y. -F. Li wrote:
> Hello,
>
> I'd appreciate it if anyone could provide information (experiences
> or publications) on the following:
>
> 1. Put a tag such as FLAG, V5, etc etc, at the N- and/or C-termini,
> in order for specific detection, but not interfering with protein
> folding/structure;
> 2. Is a linker between the tag and target protein needed? What
> linkers (length, specific sequences) would you suggest?
> 3. What are the choices of such tags and why would you recommend them?
>
> Thank you for your time and sharing in advance.
>
> Yongfu Li
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