Problem with subcellular fractionation - (Nov/29/2012 )
I recently began using the subcellular fractionation kit provided by Thermo scientific (http://www.piercenet.com/browse.cfm?fldID=33BA6DF4-5056-8A76-4E23-CD7046C43DF5) and my western blots showed protein in all of the fractions where I expected only protein in the membrane and nuclear fractions. I also used anti-Lamin as a nuclear marker which showed up in all fractions.
Does anyone have experience with this kit and its general efficiency in seperation and assuming this bad seperation is not due to flaw in my technique, could you please help me to determine why my fractionation isnt so good? Also if you have found certain modifications to their protocol which improve efficiency?
Thank you very much.
This is the protocol:
If you see it in all fraction then I think CEB incubation for 10 min is very long. make it 1 min and try again.