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


  • Please log in to reply
1 reply to this topic

#1 Filippo

Filippo

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 30 September 2002 - 12:48 AM

Hello!
I have been starting doing Westerns for 6 months now, and I still have a recurrent problem I would really appreciate if anybody could help me to solve. I am transfering my proteins onto PVDF membranes with a semi-dry blotting method, and I always have a decreased efficiency of transfer on the samples situated on the edges of my gel.
Briefly, the procedure used is the following:

-rehydrate PVDF in MeOH during 2 minutes
-incubate PVDF and polyacrylamide gel into a solution of 32ml TBS 10x, 80ml MeOH and completed up to 400ml with distilled water. Incubation time 15 minutes.
-apply one blot paper (also soaked in the previously mentioned solution), then PVDF, then gel, then blot paper.
-Transfer 1hr at 15V constant. I also apply some ice on top of the transfer device to prevent overheating.

Has anyone got the same problems? Has anyone found a solution?

THANKS A LOT!!!
Filippo

#2 bioguider

bioguider

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 04 October 2002 - 09:07 AM

hi,

Add the height of the paper up and down the PVDF to be sure that the transfer efficiency is enough.




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

©1999-2013 Protocol Online, All rights reserved.