longer running of proteins in SDS PAGE! - (Oct/10/2011 )
I have 3 bands of marker close to each other, and I need to do 3 westernblot on the same filter, so, I tried to run longer the gel in order to separate well the bands,to further do western blot and have separate bands .
It seems that I lost one band, the lower one on which I should do actin W.B.
Is there possibility to perform anyway the western blot even the marker band of 42kd is lost during running! Or i need to do 2 western blot on that filter and run another gel for 3 appropriate W.B.
thanks for help
You can do all three on the same membrane, especially if you are confident about your antibodies being specific, but it would be best to ensure that you don't run the actin off the gel next time.
As for this time, I would say that you may well have lost the actin, so you will probably need to run another gel, in which case you could probe that one for all three.
Which % of acrylamyde are you using? You can try to change this in order to separate the three bands and not loosing one of them.