stripping and western blot?

caro c.paulus at NOSPAMibmc.u-strasbg.fr
Tue Aug 27 08:22:07 EST 2002


dans l'article 12434ec2.0208230543.243c2b77 at posting.google.com, leezuyoug à
leezuyoug at yahoo.com a écrit le 23/08/02 15:43 :

> Hi, I have met some problems in my stripping of PVDF membrane. I use
> the protocol as follows:  50ml 62.5mM Tris-HCl (62.5), 2% SDS, 100mM
> 2-mecaptoethanol under 50C for 30 minutes. After stripping, wash 6x 5
> minutes, then reprob again. But it is always blank without any
> signal!!!
> I am working at p38 which is one kind of MAPK, using the ECL
> reagent system.  Is there anyone can help me about this?
> thinks a lot!!!

Hi, 
I often use the same protocol to strip my membranes (I even wash only two or
three times and raise sometimes the temperature until 60-65°C).
Are you doing this in a water bath? I put 2 flasks on a magnetic heater-
strirrer, one with water and the second with the membrane and the stripping
buffer.
Do you block again the membrane before applying the antibodies?
With this method I can strip the membrane more than 5 times (with different
primary antibodies)
Hope it helps !
Bye.




More information about the Proteins mailing list