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
- - - - -

consistent expression of overlarge proteins

bacterial expression

  • Please log in to reply
1 reply to this topic

#1 ocram

ocram

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 02 November 2015 - 08:00 AM

I have recently joined a new lab and started to express bacterial recombinant proteins again.  I have expressed three, all in the same vector, a modified pET vector with simple His tag in BL21(DE3) and all three proteins are too large, by between 5 -15kDa.  The sequences are fine, (both of the empty vector and recombinants) their mobility is not affected by running on Tris-glycine or Tricine gels, but I am able to purify them on HisTrap and they give a signal with anti-His monoclonal on Western.  The problem is I am not able to further purify them either by IEX or size exclusion and their size bothers me.  How can I be sure these are really my proteins - any suggestions..??



#2 labtastic

labtastic

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 174 posts
14
Good

Posted 02 November 2015 - 08:12 AM

Are you saying the proteins migrate on SDS-PAGE gels ~5-15kDa larger than their predicted size? If so, I wouldn't worry about that. SDS-PAGE is not always a great indicator of protein size...it is not unusual for things to run faster or slower than expected. 

 

If you want to be absolutely sure these are your proteins, run them out on a gel, cut the bands out and send for MS/MS sequencing. Or, if you have a specific activity assay of some kind then you can do that of course.

 

Why can't you do ion exchange or size exclusion? If you explain more then we might be able to help troubleshoot more effectively.


Edited by labtastic, 02 November 2015 - 09:13 AM.





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

©1999-2013 Protocol Online, All rights reserved.