Purifying nuclear fraction from skeletal muscle homogenate - (May/04/2009 )

I am trying to isolate the nuclear fraction of whole skeletal muscle homogenates. I am aware that there are commercial kits available, but since I have a limited amount of tissue, I am trying to work from our current protocol to see if I can get a little more data out of each sample.

Our current protol:
We homogenize each sample on high in a buffer that also contains 1% Tritonx100. We let the samples sit on ice for 1 hour and then centrifuge at 13,000G for 30mins. The supernatant is collected and labled as the cytosolic and membrane bound protein fraction which is used for western blotting. I am assuming the the pellet contains myofibrillar, connective tissue, and possibly mitchondria and nuclear fraction (?).

My first question is if my assumptions are correct on what is found in each fraction? Second, is there a protocol to purify the nuclear proteins from the pellet?

You had better to use Sucrose gradient centrifugation to get your specific fraction. There are many protocols around. Hope it will help.

James
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