I want to strip the protein off PVDF membrane - (Oct/11/2006 )
Does anybody know of a buffer that WILL strip off the protein from my membrane quickly at room temperature, but will not damage the protein (assuming the transfer did not damage it already?). By damage, I mean breaking the protein or altering it's molecular weight.
-wierdpixel-
QUOTE (wierdpixel @ Oct 11 2006, 03:38 AM)
Does anybody know of a buffer that WILL strip off the protein from my membrane quickly at room temperature, but will not damage the protein (assuming the transfer did not damage it already?). By damage, I mean breaking the protein or altering it's molecular weight.
ab stripping buffer:
62.5mmol/l Tris PH6.7
100mmol/l beta-mercaptoethanol
2%SDS
-leolee2046-
QUOTE (leolee2046 @ Nov 6 2006, 10:25 AM)
QUOTE (wierdpixel @ Oct 11 2006, 03:38 AM)
Does anybody know of a buffer that WILL strip off the protein from my membrane quickly at room temperature, but will not damage the protein (assuming the transfer did not damage it already?). By damage, I mean breaking the protein or altering it's molecular weight.
ab stripping buffer:
62.5mmol/l Tris PH6.7
100mmol/l beta-mercaptoethanol
2%SDS
and incubate at 50°C for 30 minutes (you can do it in a water-bath)
very efficient, but still you might reduce the signal of the second blotting.
It's the only efficient stripping protocol I know
-Missele-
QUOTE (Missele @ Nov 6 2006, 06:15 PM)
QUOTE (leolee2046 @ Nov 6 2006, 10:25 AM)
QUOTE (wierdpixel @ Oct 11 2006, 03:38 AM)
Does anybody know of a buffer that WILL strip off the protein from my membrane quickly at room temperature, but will not damage the protein (assuming the transfer did not damage it already?). By damage, I mean breaking the protein or altering it's molecular weight.
ab stripping buffer:
62.5mmol/l Tris PH6.7
100mmol/l beta-mercaptoethanol
2%SDS
and incubate at 50°C for 30 minutes (you can do it in a water-bath)
very efficient, but still you might reduce the signal of the second blotting.
It's the only efficient stripping protocol I know
plus during incubation you should shake the blots;
another popular protocol is 20 mM glycine pH 2.0, or ready-to-use stripping solutions of various companies
by the way, I guess that the stripping buffer of Pierce contains DMSO which I do not know as an commonly ingredient in stripping solutions; but I am not sure...
-The Bearer-
QUOTE (wierdpixel @ Oct 11 2006, 04:38 AM)
Does anybody know of a buffer that WILL strip off the protein from my membrane quickly at room temperature, but will not damage the protein (assuming the transfer did not damage it already?). By damage, I mean breaking the protein or altering it's molecular weight.
are you asking for a method to remove the antibodies so that you can reprobe? or are you asking how to remove your protein from the membrane? if so, has the membrane been blocked (and/or probed)?
personally, i've only done that in conjunction with a peptide sequencer and that was destructive so it probably wouldn't suit your apparent needs.
-mdfenko-