SDS-PAGE sample preparation - (Nov/23/2008 )
Most protocols tell you to heat the protein fraction with the sample buffer (SDS, B-mercaptoethanol, BPB) in a boiling water bath for about 8 minutes. But the lab I am in right now, uses a heating mantle to heat the sample at 100 degree. Is this ok? Will it make a difference? I don't think it does but then I was wondering. Thank you!
We use in our lab both, a thermoblock and a water bath. Basicly I prefer the thermoblock, since the samples can't "go swimming". The way of heating the sample doesn't really matter. Also the temperature 80-100 isn't really important, as long as you don't evaporate all the water at the bottom of your eppi. You want to denaturate your protein. That is the important part.
I've always boiled my samples for 5 minutes at 95ºC before doing SDS-PAGE and had no problem. I also prefer the thermoblock. Recently I've read here it's enough 65ºC for 10 minutes in order to denaturate the protein. I'm trying this week.
the samples should tightly be mantled during heating then the slope of heating will be ok