After taken out from transferring, I wash the PVDF membrane with 1X TBST for 5 minutes before staining it with used or new Ponceau S. I then rinse it with dH2O to get a clear background. However, I never get defined lanes of Ponceau staining on PVDF membrane, with either used or new Ponceau solution. When I tried the same procedure on nitrocellulose membrane, it always comes out nicely. But since at the end, PVDF gives me better results, I have to use it. But I need your help to get better ponceau staining. Do you have the similar problem? What do you do?
Please look at the attached picture for better illustration. The gels were run and transferred at the same time, under the same condition, stained in the same condition. And I am sure that there is no problem with the transfer on PVDF membrane, only the stain doesn't seem to stick for some reason.
Hello Hu Hule
Ponceau S does not work well on PVDF membranes with the conventional protocol. You need to pre-wet the PVDF membrane with 20% methanol for 1-2 mins prior to Ponceau S staining because areas of PVDF which are coated with transferred protein are capable of wetting out in 20% methanol while the surrounding areas of PVDF will not. Those areas where the PVDF wets becomes optically transparent and allows visualization of protein bands. Also, use freshly prepared Ponceau S.
I hope this helps.
Best,
Hi Hu Hule
Yes, that is right. At the final step, destain in distilled water until the bands are visible.
An interesting suggestion about the methanol, is that published anywhere? Did it fix the problem Hu? We use the stain free method because it seems to be most accurate.
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