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
- - - - -

Baculovirus system -swap tag


  • Please log in to reply
No replies to this topic

#1 AK123

AK123

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 19 February 2009 - 02:00 PM

Hi,

I am currently using the pAcSecG2T vector (BD BioSciences). This is a transfer vector containing gp67 signal sequence at the N-terminus, followed by GST-tag, then the protein of interest. My previous attempt to express a protein using this system was successful. However, for some reason, the GST-fusion protein does NOT bind to my glutathione column. I suspected that the GST tag is somehow affecting the proper folding/conformation of my protein. Therefore, I tried to replace the GST tag with a 10xHis tag. Now, using this newly constructed transfer vector (sequence verified), I did the transfection again. However now, I could not detect the production of His tagged protein. I have attempted the transfection twice... still no protein detected in tranfection (media collected 4 days post transfection) by western with anti-His antibody.

I am wondering if anyone has attempted to do this & has success. I can't think of any reason why this should not work...




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

©1999-2013 Protocol Online, All rights reserved.