Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Protein purification


  • Please log in to reply
3 replies to this topic

#1 pattu

pattu

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 21 February 2005 - 08:23 AM

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

#2 sharath

sharath

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 60 posts
0
Neutral

Posted 21 February 2005 - 10:48 PM

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
Sharath B.

#3 pattu

pattu

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 22 February 2005 - 06:21 AM

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

#4 Linnea

Linnea

    member

  • Members
  • Pip
  • 5 posts
0
Neutral

Posted 21 March 2005 - 06:36 AM

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.





Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.