stripping the blot - (Apr/03/2007 )
Hello everyone,
i tried using Restore buffer from Pierce for stipping my blot. thre protocol that I followed was, washing the membrane 3 time with TBS/T followed by incubation with stripping buffer for 10 mins with stripping buffer, then washing again with TBS/T 3x 5mins. Unfortunately I found that I lost all my protein ( absolutely nno bands on ECL detection ).
Does anyone has a better stripping protocol form western blots?
Thanks
I'm simply using NAOH 0.25M. Try 5min first, wash and check with the secondary AB if the signal is completely lost. If not, strip again a little longer. Some people in my lab strip up to 30min and still have some protein left, but this will vary from protein to protein. If the background gets to high you might have to block the membrane again. Hope this helps.
There are here some useful discussions about this problem. Hope this helps you:
http://www.protocol-online.org/forums/inde...mp;hl=stripping
http://www.protocol-online.org/forums/inde...mp;hl=stripping
http://www.protocol-online.org/forums/inde...mp;hl=stripping
http://www.protocol-online.org/forums/inde...mp;hl=stripping
i tried using Restore buffer from Pierce for stipping my blot. thre protocol that I followed was, washing the membrane 3 time with TBS/T followed by incubation with stripping buffer for 10 mins with stripping buffer, then washing again with TBS/T 3x 5mins. Unfortunately I found that I lost all my protein ( absolutely nno bands on ECL detection ).
Does anyone has a better stripping protocol form western blots?
Thanks
we use Restore stripping buffer from Pierce routinely and so far we are not faced with this kind of problem; you have to wash generously to wash all stripping buffer away to avoid effects on antibody re-probing
I had the opposite problem : no stripping with the second bottle we bought, while it was working perfectly with the first one.
so, now I make my own buffer, with tris, beta-mercaptoethanol and SDS. Works fine.
you have to wash a lot.
so, now I make my own buffer, with tris, beta-mercaptoethanol and SDS. Works fine.
you have to wash a lot.
so different lots of Pierce Restore buffer had different effects? did you complain about your problem at Pierce? what did they say?
so, now I make my own buffer, with tris, beta-mercaptoethanol and SDS. Works fine.
you have to wash a lot.
so different lots of Pierce Restore buffer had different effects? did you complain about your problem at Pierce? what did they say?
I did.
I don't remember exactly what they said, they sent a third bottle for free, but it was not better. I was surprised that it was no more working at 100% because I was trying to strip the same antibody.
So I prefer to prepare my own buffer.
However, I don't complain too much, because Pierce offers some other products that work fine.