 Western blot - Stripping and Reprobing |
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 Mar 4 2005, 12:21 PM
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#1
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member  Group: Active Members Posts: 14 Joined: 18-January 05 Member No.: 5383  |
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 |
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Mar 4 2005, 05:41 PM
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#2
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Enthusiast Group: Active Members Posts: 80 Joined: 17-November 04 Member No.: 4791 |
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). |
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Mar 7 2005, 12:51 AM
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#3
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Veteran Group: Active Members Posts: 291 Joined: 15-April 04 From: Bordeaux - France Member No.: 2987 |
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 |
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Mar 7 2005, 06:41 AM
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#4
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member Group: Active Members Posts: 7 Joined: 3-March 05 Member No.: 5928 |
Same protocole as Fred33! Confirme the membrane is ready to use!
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Mar 8 2005, 06:36 AM
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#5
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Enthusiast Group: Active Members Posts: 45 Joined: 16-July 04 Member No.: 3526 |
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 |
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Mar 15 2005, 09:11 PM
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#6
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Enthusiast Group: Active Members Posts: 35 Joined: 8-March 05 From: Western University, CHRI, Canada Member No.: 5999 |
QUOTE (cwong1215 @ Mar 4 2005, 01:21 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 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? |
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Mar 17 2005, 06:36 AM
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#7
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member Group: Active Members Posts: 10 Joined: 26-January 05 Member No.: 5469 |
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. |
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Mar 17 2005, 06:45 AM
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#8
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Enthusiast Group: Active Members Posts: 95 Joined: 13-December 04 From: Barcelona Member No.: 5058 |
I use the same protocol as patirl. Smells bad but works good.
Cheers 
-------------------- Science is a wonderful thing if one does not have to earn one's living at it
(A.Einstein) |
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Mar 21 2005, 11:40 AM
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#9
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Enthusiast Group: Active Members Posts: 35 Joined: 8-March 05 From: Western University, CHRI, Canada Member No.: 5999 |
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 
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