IUBio Biosequences .. Software .. Molbio soft .. Network News .. FTP

yeast homogenates

Chris Doering cdoerin at emory.edu
Tue May 19 11:58:16 EST 1998


klennon at ACS1.BU.EDU wrote:
> 
> Hi.
> 
> 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!
> 
> Sincerely,
> 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!

Good luck,

Brett Burkholder
Dean Danner Lab
Emory University
Department of Genetics
Atlanta, GA 30322




More information about the Yeast mailing list

Send comments to us at biosci-help [At] net.bio.net