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
- - - - -

Help with small protein with his-tag purification


  • Please log in to reply
7 replies to this topic

#1 Dianarodriguez

Dianarodriguez

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 03 August 2011 - 04:31 AM

hi,
i have a small protein with his-tag and tevsite. i have very nice expression but the problem is,that the protein no binding in to the Ni-NTA. the protein is in the soluble fraction. i trie many buffer and too witout imidazol but no work , i trie to in GdmCl 6M and no work, because for me is no importan if my amilin peptid 37 aa. is folding or no , but no work

#2 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,785 posts
132
Excellent

Posted 03 August 2011 - 11:51 AM

try 8M urea
talent does what it can
genius does what it must
i do what i get paid to do

#3 Papaver

Papaver

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 77 posts
19
Good

Posted 10 August 2011 - 05:59 AM

Have you checked if the his tag is in frame after cloning. Is the His tag N-terminal? If so, you can try cloning it C-terminal (or vice versa).

#4 gorkin

gorkin

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 16 August 2011 - 08:59 PM

Dont use too much resin, i.e. histrap binds around 20-40 mg/ml. Try changing the Ni-NTA to Co-Talon or other metals with Talon. Whats your pH. I had similar problems with a protein when I had a 12xHis tag at the N-terminus (membrane protein). In this case my conclusion was that the tag was bound to something like a lipid when I purified in the presence of certain detergents which did not remove all of the tightly bound lipids. Your situation might be similar, i.e. a protein binds very tightly and occludes the tag.

#5 prodes

prodes

    member

  • Active Members
  • Pip
  • 25 posts
1
Neutral

Posted 18 August 2011 - 07:48 PM

Do you use reducing agent? NiNTA is not compatible with concentrations of DTT above 1mM and b-ME above 10mM.

Such a small protein/peptide is likely to be digested by proteases as well. Have you done western to confirm the expression and the protein's presence in the soluble fraction? I would consider changing the expression if you do not see it on the western probably by fusing it to MBP or so if you do E. coli over-expression.

#6 CAT

CAT

    member

  • Active Members
  • Pip
  • 24 posts
0
Neutral

Posted 16 February 2012 - 01:39 PM

I had the same problem. Mine was 10x his tagged to the furthest N-terminus of a membrane protein. I checked the expression level with western blot and binding experiments. I blamed that the N- 10X his were wrapped and therefore, not accessible to the resin. Is this reasoning valid? also wonder how to solve it?

#7 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,471 posts
247
Excellent

Posted 16 February 2012 - 05:50 PM

N terminal tagging of membrane proteins is problematic. They often include an export tag which is cleaved, and the remaining C terminal fragment is lipidated to bind to the membrane. If your tag is on the N terminus, it likely is cleaved as well.

#8 Bluefunk

Bluefunk

    member

  • Active Members
  • Pip
  • 20 posts
0
Neutral

Posted 16 February 2012 - 07:55 PM

I would ensure your protein is being expressed first and your his tag is there by anti-His western blot




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

©1999-2013 Protocol Online, All rights reserved.