I wonder if anybody met this situation before..
I ran a gel electrophoresis at 180v for 50 mins and do transfer in 20v 300mA 90mins. after transfering my protein to PVDF membrane, the ladder on the PVDF became all in red color (every band of them ) ...
I don't know what's this supposed to mean ? should I feel worried ? and is my protein successfully being transfer to the membrane ?
it could be a pH effect on the dye molecule of the standards but the standards transferred so I would expect that the proteins in your samples have also transferred correctly
your migration and transfer conditions are so intense. I suppose that the gel was warm after your migration and transfer. High voltage results in high temperature which may cause some problems.
Anyway, you can simply color your membrane with Ponceau Red and if you see bands, then your proteins are transferred; but you may have difficulty to obtain "beautiful" bands, because your WB conditions are not optimized.
Usually, I perform my migration at 75 V for 25 min, then at 100 V (choose constant voltage option). Your can adapt the time of migration, depending on the% of your gel and size of your proteins. For transfer, I usually perform liquid transfer, use a constant intensity and apply 120mA per 1 gel (if your have 2 gels 240mA and So). Certainly, it will take more time but you are at least sure to have reproducible results and good blots for future publications.
Best,
it could be a pH effect on the dye molecule of the standards but the standards transferred so I would expect that the proteins in your samples have also transferred correctly
thanks for your suggestion !!
I color my gel with coomassie blue earlier today , i think my protein only partially transfer to the PVDF membrane. (oh and I wet my PVDF with methanol in advance ;)
I will change some of transfer condition next time !! really thanks a lot to all of you !! :)))
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