3 replies to this topic

[pattu]

Hi,
I have a few His-tag proteins expressed in insect cells using baculovirus. Everytime I purify these proteins its stays good and purifies well untill I reach the final Gel filtration step. Under all the conditions I have tried (Different conc of NaCl, Glycerol etc.) I donot get any elution of my protein from the column. As if it disappears. I use a Superdex 200 HR 10/30 column and tried several conditions.
Is it known that His Tag proteins stick to the columns or spread over large elution volums so that they can be seen?
If anyone has faced same problem and figured out a way please help me.
Thanks
Pattu

[sharath]

why do you use gel filtration with an his-tag protein. the standard method is to employ affinity chromatography with nickel sepharose.

Sharath.B
Research Fellow
National Chemical Laboratory,
India

[pattu]

Hi Sharath,
I do use Ni-Affinity followed by ion exchange. But the problem I am talking about occurs at the final step of purification using gel filtration.
Thanks

[Linnea]

Hi,
I'm purifying a His-tagged protein using a Ni-NTA column followed by a Superdex 75 10/300 tricorn column. I used 6 M GuHCl as mobile phase as my protein is very prone to aggregate. My protein was eluted nice and easily. Can't say why yours isn't, just wanted to tell you that it can be done.

Have you used the SEC-equipment before? Are you sure that your loading procedure is working properly?

Edited by Linnea, 21 March 2005 - 06:36 AM.