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!
- - - - -

TNT coupled in vitro translation

  • Please log in to reply
No replies to this topic

#1 saad



  • Active Members
  • PipPipPipPipPip
  • 49 posts

Posted 01 June 2009 - 12:56 AM

Hi everyone

I need a help, actually I've done in vitro translation by using TNT in vitro couple transcription/translation with plasmid that is prepared inhome by miniprep.
- I use 35S Met to label proteins (prey and bait)
- incubation at 30C for 2 hr
- incubate the flag-protein with anti-flag beads first for 2 hr then incubate with bait protein o/n at 4C, washing 6x . add 20 ul 4X SDS heat 10 min at 70 C then load 15-20 ul on SDS-PAGE. I expose the gel using Phosphor Screen then scan with Typhoon.
I've problem with unexpected bands and some strange results and I hope to optimise this experirment.
for instance how much volume of beads"difficult to adiust the volume of beads", I use usually 25-30 ul then start equilibrate &washing
how much volume should I load on the gel?
any suggestion welcome and I appreciate every comment.


Edited by saad, 01 June 2009 - 12:59 AM.

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

©1999-2013 Protocol Online, All rights reserved.