frationation and changes localisation of human cancer lines - (Jun/29/2007 )

Dear
I trying to localize, the changes in one plasma membrane protein of human cancer cell line. First I lyse my cells in swelling buffer and I fractione my cells: I centrifuge the lysate at 800g for 10 minutes, use the supernant and centrifuge for 20000g at 15 minutes, my pellet I guess suppose to contain mitochondria fraction. then I do further centrifugation at 100 000g for one hour where my pellet has the the plasma membrane protein in the microsomal fraction, I want to see changes and the supernant is the cytosolique fraction
However my western, showed a strong signal in the mitchongrial inside microsomal fraction, that should happen because the plasma membrane protein.
I would like you to help me, may be my fractionation is not correct and I still have plasma membrane protein in the microsomal fraction. Can you give me proper method to do so because all the literature seems unclear.
Thank you
Jean Aymard ( Amsterdam)

can you give more detail sucrose debsity gradient please
may be I was not clear : here is what I did:
lysate----> centrifugation 800g at 10 min discard pellet, supernant----> centrifugation 20000g at 15 min: pellet I though was enriched with mitochondria and supernant----> 100 000g : pellet plasma membrane there I thought and suprnant is cytosol but my western blot give me strong signal for I what I thought mitochondrail fraction forlocalisation of plasma membrane protein
Thanks
Jean Aymard

Your 20Kg pellet contains PM amd other big membrane fragments, so of course when you do WB you will find PM proteins there. The 100Kg step spin of supernatant would pelllet other membrane bound compartments like ER, Golgi, endosomes...

Check David James paper (GLUT4 studies) or search in PubMed for cell fractionation.

For us, we do as followed:
- Homogenize cells in cold HES buffer ( 20mM Hepes, pH 7.4, 1mM EDTA, 250mM sucrose)
- Spin 17.3Kg, 20min, 4oC (Prior to that can try the 800g step to get rid of debris)
- The pellet is washed and resuspended in 1ml HES. Layer this on top of a 1.12M sucrose cushion and spin at 100Kg, 1h, 4oC (so the 100Kg should be for the PELLET and NOT the supernatant). PM is found in the membrane layer above the cushion.
- Small fragments of PM could still be found in the supernatant (of the 17.3 Kg step), but the majority should be in the above step.

Thanks I will try that tomorrow