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!
- - - - -

Protein Extraction: Extremely Sticky e coli lysate

  • Please log in to reply
1 reply to this topic

#1 qpwoei4756



  • Active Members
  • Pip
  • 6 posts

Posted 04 December 2012 - 04:09 PM


I'm trying to extract a 6x his tagged protein from BL21 competent cells, but the pellets are so sticky I can't collect my protein.
What I did was induce my protein using IPTG for 4hr, pelleted them and placed it in -20C. So far everything's fine.
When I took it out three days later and thawed it, however, it looked sticky just by shaking the tubes.
They are sticky as snout and no matter how much I mix it with my lysis buffer, it doesn't dissolve.
I tried adding DNAse, sonicating for a longer time, vortexing, and centrifuging them at twice the normal speed, but the stickiness won't go away. I can't get the supernatant, where my protein would have been located.

Why is this happening? Should I try to collect my proteins without ever freezing the cell pellets? Thank You.

#2 PhDinAcronyms



  • Active Members
  • PipPipPipPipPip
  • 37 posts

Posted 05 December 2012 - 03:54 PM

If the stickiness happened after lysis, I would think it's DNA. Did you also add MgCl2 with the DNase? Needs magnesium to work

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

©1999-2013 Protocol Online, All rights reserved.