 Western blot stripping problem |
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  Jan 26 2005, 09:44 AM
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#1
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member  Group: Active Members Posts: 6 Joined: 6-December 04 From: UFSC, Brazil Member No.: 4995  |
Hi! I have stained with sucess a protein with 27 KDa, but when I tried use the membrane again to stain other protein, I observed the last bands.
How can I remove Ab 1st stained from my membranes? I have ised strip with NaOH 0.1M. Thanks |
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Jan 26 2005, 10:31 AM
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#2
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Enthusiast Group: Active Members Posts: 95 Joined: 13-December 04 From: Barcelona Member No.: 5058 |
You can try 62.5 mM Tris pH 6.8, 2% SDS and 100 mM 2-mercaptoethanol. Incubate the membrane at 50ºC for 30 min with ocassional agitation and then wash with TBST and block again.
-------------------- Science is a wonderful thing if one does not have to earn one's living at it
(A.Einstein) |
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Jan 28 2005, 10:04 AM
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#3
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member Group: Active Members Posts: 6 Joined: 28-January 05 Member No.: 5498 |
5 min H2O
5-10 min 0.2 H NaOH 5 min H2O Works good. |
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Mar 31 2005, 05:30 AM
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#4
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member Group: Members Posts: 3 Joined: 31-March 05 Member No.: 6359 |
Hi all,
Usually, after I do the 1st stripping, all most proteins in the membrane disappeared ( I checked it by staining that membrane with Ponceau solution) or just remained a little.  The contents of stripping buffer I used is: + 0.2 M Glycine (pH 2.5) + 0.05% Tween 20 I incubated the membrane at 80oC for 30 minutes. And then, I washed it 10 min x 3 times with TBST before blocking with skim milk. What's wrong in my method and how can I solve the problem? Thank you! 
This post has been edited by Gin: Mar 31 2005, 05:37 AM |
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Mar 31 2005, 06:21 AM
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#5
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Veteran Group: Active Members Posts: 291 Joined: 15-April 04 From: Bordeaux - France Member No.: 2987 |
hi
i start to see same problems as you and i use the same protocol of stripping. Hence i'm wondering is "our" glycine too old? mine is more than 3 month... but for stripping protocol, there was a discussion here from protein and proteomics forum fred |
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Mar 31 2005, 06:48 AM
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#6
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member Group: Members Posts: 3 Joined: 31-March 05 Member No.: 6359 |
QUOTE (fred_33 @ Apr 1 2005, 12:21 AM) hi i start to see same problems as you and i use the same protocol of stripping. Hence i'm wondering is "our" glycine too old? mine is more than 3 month... Hi Fred, Thanks for your reply. Well, but I don't think so. Because another person who taught me that method, he also used the same Glycine with me. And of course, no matter  Thus, I considered the pH of buffer, or the incubate-temperature, or the shaking... are they important to the result of stripping?When you had that problem, how can you overcome it, Fred? You changed with "new" glycine? Gin, |
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Mar 31 2005, 07:58 AM
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#7
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Veteran Group: Active Members Posts: 291 Joined: 15-April 04 From: Bordeaux - France Member No.: 2987 |
hi
i think ph is not that important caus i use pH 2.7 at 80°C for 30' but the two last stripping were not by shaking i must admit, and the residual bands apperead just in the pas times. hence i assume that shaking would increase efficiency |
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