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
- - - - -

How can I get protein in soluble fration


  • Please log in to reply
2 replies to this topic

#1 saad

saad

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 49 posts
0
Neutral

Posted 11 September 2010 - 02:47 AM

Hi all,

I have cloned tow fragments ( 30 and 24 KDa)from the same protein (human)into 3 different vetors ( pGEX, pMAL and pHUE) with 3 different tags ( GST, MBP and HIS6-Ubiquitin tags). I need to purify them in native condition. I did a trail expression in E. coli ( BL21 and Rosseta) at 37C and 20 degree with 0.4 mM IPTG. In all cases, all the six construts were in an insoluble fraction and almost nothing is in the supernatant.

Could you please help me to make these proteins soluble .

regards,

#2 Aris

Aris

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 67 posts
0
Neutral

Posted 11 September 2010 - 09:58 AM

Hi
I am not sure if this would help but I know that some proteins, in particular certain enzymes, can be solubilised using high concentrations of Urea (i.e. 4-6 M Urea). In this way you can get them out of tissues in their native form. Still, you will need to purify your protein afterwards using dialysis.

#3 protolder

protolder

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 293 posts
9
Neutral

Posted 12 September 2010 - 09:53 PM

Hola, I have answered before a similar question. The fact is that if any of both strains isn´t the subtype lacIq probably you have a quasi constitutive expresion in LB or other mediums without glucose.Have you analyze t=0 of induction ?. In order to facilitate a light expression in my lab grow the cultures at 25 ēC. Good luck




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

©1999-2013 Protocol Online, All rights reserved.