Hi Amala,
1. Did you verify the sequence or the presence of the His-tag? If you were
not the person for cloning. I used to spend 14 months working on an clone
and eventually I was allowed to check the sequence and verify the
expression of His-tag by Western-Blot. There was no his-tag.
2. You can t
Dear Amala
Please increase NaCl concentration to 200mM from 50mM. That can help you
out by increasing affinity of your protein to bead and will delay the
elution time.
On Mon, Aug 13, 2018, 7:20 PM amala mathimaran wrote:
> Dear All
>
> I am working with HIS – tag protein in N-terminal (hexa hi
Hello Amala,
Usually Ni-NTA won't have this kind of problem of binding. Probably your
protein is have interaction with hexagon his tag which is affecting its
affinity towards beads. You can try putting tag in C- terminal end of the
protein.
On Mon, 13 Aug 2018, 8:02 pm Artem Evdokimov,
wrote:
Hi Amala,
Depending on the resin you used there may be a conflict with BME and also
50 mM TRIS may be too much at the pH 7.5. Easy to test: use HEPES pH 7.5
and TCEP instead of BME, or use 30 mM TRIS at pH 8.0
Alternatively your protein is aggregated and does not bind well...
Artem
- Cosmic Cat
Dear All
I am working with HIS – tag protein in N-terminal (hexa histidine). The
protein from Prokaryotic origin cloned into pET30a+ vector and
expressed in *E.coli
*BL21 cells. The expression was good. I am trying to purify a protein using
Ni-affinity column equilibrate with Buffer A 50mM Tris pH
