klennon at ACS1.BU.EDU wrote:
>> I am looking for a protocol to homogenize yeast cells for Western blot
> analyses that does not involve using a bead beater. I just went through a
> lovely protocol using glass beads, only to have my cells
> completely intact at the end! I have routinely done RNA isolations from yeast
> using glass beads, so I know you have to vortex the hell out of them to break
> them, but it didn't work well at all with this protocol.
>> Any help would be greatly appreciated!
> Kelley Lennon, D.Sc.
Try spheroplasting the yeast first. I have increased my yield of
mitochondria from yeast almost 10-fold by spheroplasting before using
the glass bead smash.
Also, make sure you use only 1 x cell volume of buffer to do the smash,
and lots of beads (I use approx. 0.75 cc of beads per mL of liquid).
6 x 30" vortexing at maximum speed will ususally lyse 70 - 90% of the
spheroplasts. And be sure to use protease inhibitors!
Dean Danner Lab
Department of Genetics
Atlanta, GA 30322