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
- - - - -

Protien expression


  • Please log in to reply
3 replies to this topic

#1 shilpamadhu

shilpamadhu

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 08 February 2005 - 07:32 AM

Hi,
I am trying to express protien, with induction of IPTG( 1mM) for 4 hours, but the expression is low. So, can I try increasing the con. What could be highest concentration of IPTG I can use??

thanks,
shilpa.

#2 Sprag

Sprag

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 45 posts
0
Neutral

Posted 08 February 2005 - 09:47 AM

I don't think the concentration of IPTG, when using 1mM, matters for increasing the expression.
Instead, try to induce at higher density (OD600 > 0.6), or induce for longer time (overnight at 30 degrees). Or use a different strain. Some strains are optimized for expression of mammalian proteins.

#3 sharath

sharath

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 60 posts
0
Neutral

Posted 21 February 2005 - 11:25 PM

from your mesage I understand that you are adding IPTG during inocluation itself. that's wrong. The correct way is to grow the cells to a mid log phase , say for 3 hrs or so and then add IPTG. 1mM IPTG is the highest concentration good enough.

If IPTG is aded in the beginning itself then the cells start expressing the protein immediately and most of the cellular resources are siphoned off and the cell division machinery is deprived.

good luck

Sharath.B
Research Fellow
National Chemical Laboratory
India
Sharath B.

#4 Simonsays

Simonsays

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 72 posts
0
Neutral

Posted 22 February 2005 - 11:35 AM

I agree with Sharath... IPTG should be added only when your bacterias are in the exponential phase of growth.

Simon




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

©1999-2013 Protocol Online, All rights reserved.