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
