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
- - - - -

His-tagged protein purification


  • Please log in to reply
3 replies to this topic

#1 Richard.21

Richard.21

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 38 posts
0
Neutral

Posted 27 December 2010 - 04:44 AM

Hi everyone!

I work with his-tagged protein (17 KDa) expressed in E. coli. I use sonicator and 8M (pH 8.0) urea to denaturate and extract my protein. After, we bind protein with Ni-NTA (Qiagen) for 2 hours in ice, and then put to Polypropylen Columns (Qiagen). Collect the Flow-Throught, then four steps washing with 8M (pH 6.3) urea followed by four steps washing with 8M (pH 5.9)urea and finally elution in four fraction with 8M (pH 4.5).
The problem is: when i run SDS-PAGe and silver staining i see contamination in my elution protein. the other problem is the little yield of protein.
Does anyone know any kind of tips to improve my purification and improve the yield of my protein.

Thank you!!

#2 protolder

protolder

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 293 posts
9
Neutral

Posted 27 December 2010 - 03:48 PM

Hola, I suposse that the denaturation of your lysate is due because your protein is insoluble, or aggregate, and you prefer elute with acidic pH than with imidazole.To clean your preparation I would sonicate in an aquous buffer to elimnate soluble proteins and after it denature with urea, centrifuge and apply the supernatant to the column. If you say that have litte yield is because you know the total amount produced and applied to the colum, so, there is protein non eluted.I donīt know if you can lowering the pH a bit more, but why dont you try to use imidazole, or moreover to try refolding inside the colum, aplying with urea washing without it and eluting with pH or imidazole. Buena suerte y feliz aņo nuevo

#3 paramyosin

paramyosin

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 34 posts
0
Neutral

Posted 29 December 2010 - 06:57 AM

Hello,
Two things: first, I would try a different binding protocolo, 2 hours in ice is not a good election, I would leave the protein binding all night long at 4šC or 1-2 hours at RT, at RT the binding is improved. Second, if you see your elution fractions dirty maybe is because you donīt wash enough in your 1 or 2š washing step. Sometimes you need to add an extra step (capture) in your purification protocolo in order to obtain a purer preparation: Q, CM, S, HIC, something with a high binding dynamic capacity...

Have luck
"Research without indebtedness is suspect, and somebody must always, somehow, be thanked." Umberto Eco

#4 Richard.21

Richard.21

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 38 posts
0
Neutral

Posted 31 December 2010 - 04:41 AM

Hi!

Thanks for everyone replying me!!

So, someone told me, to avoid excess incubation with Ni-NTA agarose because unespecific proteins could bind in Ni-NTA and it will be eluted in my fractions. What do you think? How long should i incubate?
so dou you recomend more washing, but too many washing steps would not unbind my protein and the final yield?
What about Polypropylen Columns, they are good for purification?
My protocol, we don't use Imidazole, what about?

Thank you in advance!!

Hello,
Two things: first, I would try a different binding protocolo, 2 hours in ice is not a good election, I would leave the protein binding all night long at 4šC or 1-2 hours at RT, at RT the binding is improved. Second, if you see your elution fractions dirty maybe is because you donīt wash enough in your 1 or 2š washing step. Sometimes you need to add an extra step (capture) in your purification protocolo in order to obtain a purer preparation: Q, CM, S, HIC, something with a high binding dynamic capacity...

Have luck






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

©1999-2013 Protocol Online, All rights reserved.