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
- - - - -

WESTERN BLOT OF LARGE AND MINORITY PROTEIN


  • Please log in to reply
1 reply to this topic

#1 Gonzo

Gonzo

    member

  • Active Members
  • Pip
  • 9 posts
0
Neutral

Posted 27 January 2010 - 02:04 PM

Hi, there!

I'm trying to detect one 260kDa protein by common western blot method. Actually I use 6% (bis)acrylamide self-made gels, make eletrophoresis for 1h30min and finally transfer to a PVDF membrane by using semi dry method. Moreover, I've made the following variations:
1.- I've tried to run from 30ug till 350ug protein (in this case, I could only observe a miser smear...)
2.- The transfer time was ranged from 15min till 2hrs at 20v.
3.- My lysis buffer consists in TRITONX100. However, I could try NP40 or SDS buffer and sonication.

Since now I failed to find any protein. Do you know some variation which could increase my method's efficiency??

Thanks!

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,666 posts
396
Excellent

Posted 27 January 2010 - 03:26 PM

Large protein - long run and long transfer would be my best suggestion. I would run at 100-120 v for several hours and then wet transfer overnight at 15 V.

Adding some SDS to your lysis buffer and running buffers will help with the run and transfer.




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

©1999-2013 Protocol Online, All rights reserved.