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
- - - - -

Dimeric form of a protein?


  • Please log in to reply
2 replies to this topic

#1 Axel

Axel

    member

  • Active Members
  • Pip
  • 7 posts
0
Neutral

Posted 18 January 2010 - 06:03 AM

Hi!
I am trying to express a protein and I'm running SDS-PAGE to determine if I have achieved anything. After a few failed tests at different temperatures and IPTG concentrations, I tried a protocol for inclusion body purification to that point that I have dissolved any inclusion bodies, and then tested the results on a gel. Now, finally I get a clear band that hints I have actually got protein, but it is roughly at double the weight of what I expected, which leads me to suspect I have some sort of dimeric form.

Well, my question is if that could be a logical conclusion? I thought that the SDS usually separates dimers, and even when I have denatured with urea they seem to form the dimer. I have twelve extra amino acids from the expression vector that I will try to remove and I hope that does the trick, otherwise I'm in need of ideas..

#2 Vini

Vini

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 178 posts
1
Neutral

Posted 18 January 2010 - 07:24 AM

Hi!
I am trying to express a protein and I'm running SDS-PAGE to determine if I have achieved anything. After a few failed tests at different temperatures and IPTG concentrations, I tried a protocol for inclusion body purification to that point that I have dissolved any inclusion bodies, and then tested the results on a gel. Now, finally I get a clear band that hints I have actually got protein, but it is roughly at double the weight of what I expected, which leads me to suspect I have some sort of dimeric form.

Well, my question is if that could be a logical conclusion? I thought that the SDS usually separates dimers, and even when I have denatured with urea they seem to form the dimer. I have twelve extra amino acids from the expression vector that I will try to remove and I hope that does the trick, otherwise I'm in need of ideas..


hey its quite possible that ur protein is actually showing anomalous mobility on the gel!. many proteins do show such profile coz of various reasons (highly acidic residues, hydrophobicity factors etc.)....i had a 27kDa protein running at 11kDa.........i think the best bet would be to confirm the identity of ur protein using western blot or mass spec.

#3 jah

jah

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 44 posts
0
Neutral

Posted 18 January 2010 - 06:34 PM

You didn't mention that you tried any reducing agents....could you have disulfide bridges?




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

©1999-2013 Protocol Online, All rights reserved.