centrifuging - protein extraction (Oct/20/2004 )

Hi,

Our lab protocol for obtaining protein from rat brain is:

1. Perfuse with saline
2. Remove brain
3. Sonicate in sample buffer for 15 sec.
4. Centrifuge at 14,000 rpm X 15 min.
5. Obtain supernatant discard solid

My question is this: During centrifuging, will transmembrane proteins, such as receptors be lost, or are they realeased during sonicating?

Thanks,

Jason

During sonication membranes break releasing the content of the cells and organells and they close again. No, you don't release transmembrane proteins, they will stay in the pellet.