lysing S. cerevisiae cells
dan at cubmol.bio.columbia.edu
Tue Jan 18 16:44:01 EST 1994
In article <1994Jan13.194555.26328 at midway.uchicago.edu>,
Debbie Nathan <dnathan at midway.uchicago.edu> wrote:
>We are having trouble making good native S. cerevisiae lysates.
>Generally (in the cold room) we beat a concentrated cell slurry with an
>equal volume of 0.45 uM glass beads for 5 x 30 sec using a hand vortexer
>set at the highest speed or for 2 min using a VWR multi vortexer. While
Do you really use 0.45 micrometer beads? Isn't that awful small? We
use beads that are about 0.5 millimeters in diameter.
Other than that I do pretty much the same thing. Here are the things
I can think of:
1) Cell density too low. I have found that you can get extremely low yields
if you don't start with enough cells. Keep them as concentrated as you can.
I might resuspend a log phase culture in 1/100 volume prior to vortexing.
2) Protease inhibitors. Someone else mentioned this. Who knows? I always
3) I grind 12-15 times for 15 seconds, with alternating to an ice bucket. I
don't do it in the cold room.
4) Follow the grinding under the microscope. You should see the cells as
ghosts or visibly broken. If the cytoplasmic debris isn't quite concentrated
(lot's of little jiggling stuff floating around) then you need more
5) Glass tubes? Typically, I vortex 1 ml with 1 ml of beads in a 15 ml
glass tube. Some people think plastic isn't hard enough to break cells.
I think they are nuts, but who can say?
6) Use log phase cells. Saturated cells can take considerable more time
to break. I think they are OK as long as you monitor them for breakage.
>we don't have much of a problem with protein degradation using this
>method, our yields of protein are quite low. When we have tried
>increasing the time we beat the cells, we get degradation. Does anyone
I don't think you should get degredation as a usual case. That might
indicate a need for protease inhibitors. If you use them, make new
solutions. They go bad. My samples are usually stable for at least 2 weeks
at 4 C.
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