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!
- - - - -

Tag proteins for CO-IP and confocal

  • Please log in to reply
1 reply to this topic

#1 nepeanbone



  • Members
  • Pip
  • 1 posts

Posted 06 May 2011 - 01:14 AM

I am planning to start some experiments for protein-protein interactions using confocal and CO-IP
I want to construct plasmids labelled with either GFP and His. I will use the His tag for IP.
I am going to use cDNA using primers with restriction site extensions to clone into plasmids. The His Tag is at the N terminus and I will also probably get a GFP plasmid tagged at the N terminus. The cDNA will be inserted inframe following the Tag, so does it need to include the start codon? I assume that it has. If the tag had to be at the C terminus and so the MCS is before it, do I have to include the inserted gene's stop codon? Or if I do the tag will not be fused with my protein?
Please advise
Thank you

#2 bob1


    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 6,707 posts

Posted 06 May 2011 - 05:21 PM

If you tag C-terminally do not include the gene's stop codon or you won't get the tag expressed.

I include the start codon at the N-terminus even if there is one on the tag. You can add a kozak sequence before the start codon to enhance the expression. This should take the form GCCACCATG, where the ATG is the start codon.

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

©1999-2013 Protocol Online, All rights reserved.