Hi,
to add to your list:
You could also try Ni-IDA (e.g. from Pharmacia, I hope I remember the name
correctly) instead of Ni-NTA. If I remember correctly, IDA chelates the
metal ion only two-fld instead of three-fold as the NTA does.
During my PhD th protein would not bind at all to NTA but it work greatly
with IDA - and I mean 'not at all' while the protein expressed at 40-60
mg/l LB.
Cheers, Tim
--
Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen
GPG Key ID = A46BEE1A
On Thu, 1 Mar 2007, Ngo Duc Tri wrote:
Dear CCP4 users,
Thank you all of your advices which help me to solve this problem. I believe
that all of your advices will help me and others having the same problem.
This is the summary of experienced advices about how you will do if the
protein having His-tag don't bind to NTA resin.
1. The protein is aggregated or misfolded in such as way as to hide the His
tag, try adding things like glycerol or detergents to try to prevent
aggregation
2. Use mono-q or mono-s as a first step. After that, running the NTA resin.
This way is helpful when dealing with insect cells.
3. Dialyze the lysate to remove contaminants that prevent binding. This is
very common for Bv expression, but somewhat unusual for EC
4. Partial unfold of protein by including 1-3 M Urea in the buffer A
5. Incubate the protein with the resin in an end-over-end nutator to
increase the interaction time or run the flow-through over a fresh batch of
resin and do this several times until getting enough protein
6. Change the tag from e.g the N-terminus to the C-terminus ? Or if you have
a structural homolog you could add the His tag into a loop, which is
exposed.
7. Introduce a linker between the protein and the his-tag or create a 8xhis
or 10xhis tag to enhance binding to the nta matrix
8. Using the Strep-II instead of His-tag
My best regards,
TriNgo
Sungkyunkwan University
