nuclear extraction of blood cells - (Feb/18/2011 )

Hi!

Here's my problem. I'm having some unexpected results in the detection of a nuclear protein. I suspect that this is related with the nuclear extraction step because, when I visualise the cells in the microscope, I see no swelling of the cells and lysis of the cytoplasmic membranes when it is supposed to happen. I have this problem in fresh human monocytes and neutrophils. So I was woundering if someone has a protocol of nuclear extraction that really works with this type of cells. Also, I would like to know if phosphatase inhibitors are essential to nuclear extraction because I've seen a protocol that uses them in the cell washing step but most protocols don't include them.

Thanks in advance.

You may want to try to use spin columns (filter cartridges)formate to isolate your protein. Cytosol and nuclear portion can be seperated pretty well. Protocol is easy and samples can be done in a few minutes.

Protease and phosphatase inhibitors may be needed depending on the nature of your protein and ho your samples are handled.