Electrophoresis - Pounceau membrane staining (Jan/13/2005 )
Hi! I have used transfer buffer (Tris/SDS/glycine/10 % methanol), but after transference I have no seen staing with Pounceau in my nitrocellulosis membrane, even I see staing with the Ab. Is there any methodology to solve this trouble?
Maybe you are not adding enough protein on your gel?!.. Ponceau is not as sensitive as WB!!!
Also check if you really use nitrocellulose and not PVDF!!..
I've had problems with ponceau stainings of PVDF and instead of the standard Sigma (0.1% ponceau, 5% acetic acid), i made my own ponceau as 0.2% ponceau in 3% TCA, and that worked.
i am afraid your PONCEAUS has been expired and need a fresh one.
in addition, don't use SDS in your transfer buffer again. it's not useful for most protein transfer.
I DO NOT USE SDS (important) and don't use methanol (optionnal) for my transfert solution, and everything is ok.
tris base 250mM
for one liter, it is 144.13g 10g and 30,28g respectively
tris base 250mM
for one liter, it is 144.13g and 30,28g respectively
If you want, you can add methanol :
10X transfert solution 100 ml
ddh2O qsp 1 liter.
For ponceau solution :
0,2% ponceau red
Yep, I had the exact same problem! I was trying to stain the PVDF’s with Ponceau, as someone else on the floor was doing, and to no avail. I was told that you take the membranes from the transfer buffer and immediately immerse them in Ponceau. It just wasn’t working.
Anyway, I started to play with it and I discovered that if you take the membrane and wash it with 100% methanol for a few seconds and after that you immerse it in Ponceau, and then you take it back to methanol, the bands begin to stain right in front of your eyes. (It’s like developing photo-film). I’m glad to see that other people are doing the same.
What I don't understand is why does it need re-hydration, as at no step during the transfer protocol is the membrane allowed to dry out? Just curious...
maybe SDS and ponceau Red are uncomatible...
By wahshing your membrane in 100% methanol, you get the majority of SDS in methanol (let say 90%). Then, when staining in ponceau red, the last 10% of SDS gets slowely in solution, that's why bands are appearing like photo devlopment. In fact, i suppose it beacause i do not use SDS and when i stain in ponceau, bands appears immediately (less than 2seconds)!
Any idea on that ?