Cell lysis - (Apr/24/2015 )

Hi all,

I hope this is the right subsection of the forum.  I was wondering what is considered the best general purpose method for lyasing cells to run the lysate on a denaturing reducing SDS-PAGE gel.  At the moment I use a variation of the RIPA buffer (150mM NaCl, 1 mM EDTA, 1mM EGTA, 50 mM Tris-HCl 7.6, 2% SDS, protease+phosphatase inhibitors) and lyase cells mechanically by sucking up and down through a 23G needle 20 times.  Is sonication considered a better method?  Would the buffer on its own be enough to disrupt the cells? Or would just a strong vortex be enough?

None of my proteins of interest require any special treatment, except one which requires SDS to remove it from the cytoskeleton (otherwise it just ends up in the cell debris pellet) and a phosphorylated protein which is why I add the phos inhibitor.

If you add RIPA buffer to your dish then let it sit on the cells for 5 minutes or so on ice before scraping, you will definitely recover soluble cytoplasmic and nuclear protein. I believe the additional sonication step will help retrieve chromatin associated protein. The sonication will definitely be more effective then the needle/syringe.