yeast homogenates

Michael J Conboy conboymj at leland.Stanford.EDU
Mon Apr 27 06:57:36 EST 1998


Hmm, I made the mistake early on of not using enough beads, but now it
works fine:

Lysis by beads for proteins:

- Spin down yeast (4000xg for 5 minutes, or whatever), in the cold.
Everything from now on happens on ice, preferably in the cold room. 

- resuspend in an excessive volume of protein buffer (minus SDS or
protease inhibitors), and spin down again.

- guesstimate the volume of packed yeast cells and resuspend in 1 volume
of protein buffer, with protease inhibitors (I avoid SDS buffers if I
don't want the extract to foam up, although they work fine for Westerns.) 
1.5 ml Eppendorf tubes work for packed cell volumes up to about 150
microliters, over that use 15 or 30 milliliter glass tubes)

- add beads until they fill up to the meniscus of the yeast suspension.
Don't skimp on beads here;  you want mostly beads with just enough buffer
to cover them.  

- tape Eppi tubes to the vortexer, or make a custom tube holder out of
foam rubber if you don't want to hold them.  Vortex on maximum for 10
bursts of 30 seconds, icing the tubes between bursts (they heat up).  I
think Eppi tubes vortex best for this while upside down, but suit
yourself. 

- Check an aliquot under the microscope to see of the cells are broken. 
If they are not, then vortex more.  When they are done, remove the
extract.  For Eppi tubes, poke a tiny hole in the bottom of the tube,
another in the lid (I use a 24 or so gauge needle), sit them in another
Eppi tube and spin in a microfuge at 500xg.  This requires some creative
disabling of the safety features on most microfuges, which keep one from
spinning with the lid open. Be careful and wear glasses or you may get hit
with a 2 gram projectile at about 100 kilometers per hour. 
  Use a pulled-out pipet to remove the extract from glass tubes.

- The 500xg spin will pellet unbroken cells.  Your extract is now ready to
use.

Mike Conboy
Cyert Lab
Stanford University



In article <klennon.511.35410613 at acs.bu.edu>,  <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.
>






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