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Grainy/spotty ponceau

western blotponceau grainy nitrocellulose

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#1 Nikoru

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Posted 31 August 2012 - 08:21 AM

My ponceaus are grainy/spotty, and I'm not sure why. Oddly, after I washed the membrane in TBST and restained with ponceau, the grainy spots were gone. I am curious if this was just something from the transfer buffer messing with the ponceau, or if it is an indication my membrane is no good.

I am using home-made 12% PAGEs, running at 100V (the buffer got really hot, could that be the problem?) for over an hour. I transfer overnight at 4oC onto nitrocellulose membrane.

My running buffer is pH8.3, and my transfer buffer (containing 10% MeOH) is pH8.38.

In the past, I have not run my PAGEs as long as I did recently, and think that could be the reason why (because the running buffer heats up).

Any suggestions or clues as to why this happened and how to avoid it would be greatly appreciated.

Thanks to everyone!

#2 ascacioc

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Posted 31 August 2012 - 09:30 AM

If you suddenly have this heating + longer running for your gels, something is wrong either with your running buffer (are your reusing it? then it might be too old) or the buffers needed for the preparation of the gels. Something produces a higher resistance in the gel. Higher resistance --> slower running (Ohm's law) & heating (Joule's law). I would redo all these buffer used for casting and running the gel.

The grainy/spotty thing on the Ponceau might be too much SDS in the gel that gets transferred on the membrane... but I wouldn't put my money on that. If you indeed have more than normal amount of SDS in your gel, also the transfer would be worse than usual.

#3 Akdor

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Posted 05 September 2012 - 12:45 PM

We had that problem in the lab and surprisingly we solved it but simply filtration of the PonceauS solution. Apparently it has the tendency to forms precipitates.





Also tagged with one or more of these keywords: western blotponceau, grainy, nitrocellulose

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