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Western blotting trouble shooting - Protein transfer (Jun/23/2006 )

I have trouble with staining the PVDF membrane with Ponceau.I can visualize the transfer with Commassie blue [irreversible] and know that transfer has occurred but these same bands are not seen with Ponceau-S. Also the prestained markers are not being tansferred[not seen] but they do not remain on the gel either.It's a strange problem as i haven't ever faced it. The transfer buffer is 192mM Glycine,25mMTris,20%methanol.I want to know the culprit!!.Is it the membrane,Ponceau or the Glycine.Can Glycine be used in a higher conc.


Which mA are you using? Are you sure your proteins aren't passing through the membrane?
If you see proteins with Comassie but not with Ponceau S, it means that probably you have too little protein for the staining with Ponceau (thatis less sensitive than Comassie). If you stained with Comassie, you can go on anyway with yuor WB, I read that it doesn't interfere with Ab detection. I would also try and put 2 membranes instead of one and stain both (paper-gel-PVDF-PVDF-paper) If your proteins are passing through the membrane, they will likely attach to the second one. What makes me think about this possibility is that you say that you don't have the marker either in the gel or on the PVDF. If this is the case, I wouldlower the mA and the V.
I use this amount of Glycine and Tris, I don't think it's because of them.


and i read in another post that the ponceau s solution goes off relatively quick
so make new solution and try again.....


if the marker doesn't stay on the gel and isn't visibel on the blot either are you sure you don't blot too long so most of your protein is blotting through the membran
so the protein level is so low that it can still be detected by coomassie but not with ponceau s which is less sensitive

so next time put another membrane over the first before the transfer and check


As far as I know there is nothing wrong in the buffer concentration you have been using, but I presume that the current applied might be very high or time given to the transfer is longer, check reducing the time or mAmps you are working on!!