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 - Stripping and Reprobing


  • Please log in to reply
8 replies to this topic

#1 cwong1215

cwong1215

    member

  • Active Members
  • Pip
  • 13 posts
0
Neutral

Posted 04 March 2005 - 12:21 PM

Hello,

I had a quick question about stripping and reprobing a PVDF membrane. I'm trying to figure out if anyone has had good experience with reprobing a membrane multiple times (ie: 3+). Also has anyone had luck reprobing the same membrane for a different protein? Not just looking at phospho versus non-phospho of the same protein.

Thanks

-C. Wong

#2 mikew

mikew

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 80 posts
0
Neutral

Posted 04 March 2005 - 05:41 PM

The Best method for stripping (period) is using diaminobenzadine. It is reported by John Reeds lab in the following journal.

Detection of multiple antigens on western blots.
Anal Biochem. 1996 May 1;236(2):221-8.

You cannot reprobe the blot using the same antibody using this method (at least in my hands).

#3 fred_33

fred_33

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 288 posts
0
Neutral

Posted 07 March 2005 - 12:51 AM

hi
i usually use 0.2Mglycine pH 2.5 and tween 0.05% (80 for 20'), rinse with TBS or PBS and the membrane is ready to use.

Fred

#4 margo33

margo33

    member

  • Active Members
  • Pip
  • 7 posts
0
Neutral

Posted 07 March 2005 - 06:41 AM

Same protocole as Fred33! Confirme the membrane is ready to use!

#5 Sprag

Sprag

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 45 posts
0
Neutral

Posted 08 March 2005 - 06:36 AM

If the different proteins are very different in size, and you are familiar with the antibodies (i.e. tried them individually on beforehand), I wouldn't bother stripping. Just use one at them time...
I use ReBlot from Chemicon and it works fine (the composition is obviously a secret...)

good luck

#6 mabusheh

mabusheh

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 35 posts
0
Neutral

Posted 15 March 2005 - 09:11 PM

Hello,

I had a quick question about stripping and reprobing a PVDF membrane.  I'm trying to figure out if anyone has had good experience with reprobing a membrane multiple times (ie: 3+).  Also has anyone had luck reprobing the same membrane for a different protein?  Not just looking at phospho versus non-phospho of the same protein.

Thanks

-C. Wong

I strip the membrane with 0.5 M NaOH for 10 minutes, then wash 2X with TBS.
block with NFmilk 4% or BSA 4%. I strip for 3X more than that, I get a high background.
never tried probing for different proteins, but I can't see why not?

#7 patirl

patirl

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 17 March 2005 - 06:36 AM

i use another buffer, 50mM tris pH6.8, 100mM b-mercapto and 2% SDS. Leave on for 30 mins at 50C and then wash off twice with TBS-T

I've probed the same blots for different antibodies, tubulin after using a phospho antibody. No problems.

#8 badcell

badcell

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 94 posts
1
Neutral

Posted 17 March 2005 - 06:45 AM

I use the same protocol as patirl. Smells bad but works good.
Cheers :rolleyes:
Science is a wonderful thing if one does not have to earn one's living at it
(A.Einstein)

#9 mabusheh

mabusheh

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 35 posts
0
Neutral

Posted 21 March 2005 - 11:40 AM

Hi
I figured out that you can strip the membrane with whatever protocol you want, and you can probe again for the same or different protein, it works fine :(




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

©1999-2013 Protocol Online, All rights reserved.