Does Glass Beads Hurt Intracellular Organelles?

Mike Chao mikechao at hamon.swmed.edu
Sun Feb 16 14:35:14 EST 1997


In article <nc1-1302971143520001 at phanes94.mc.duke.edu>, nc1 at acpub.duke.edu
(Namjin Chung) wrote:

>Hi,
>
>I'm interested in fractionating yeast cells, mainly vacuolar fraction and
>nuclear fraction.  
>
>When I break yeast cells, I generally use acid-washed glass beads
>(425-600u) to disrupt cells with alternating maximum vortexing for 30s and
>sitting on ice for 30s for 10-12 times.  I'm afraid if this method could
>break vacuolar and/or nuclear membranes in addition to plasma membranes
>and cell walls.
>
>Can I specifically remove nuclear fractions without contamination by any
>relatively easy method?  Can I pull out vacuolar fraction without losing
>its content?
>
>TIA
>
>
Our lab routinely prepares mitochondria using glass beads, and since
vacuoles and nuclei seem to be much bigger than mito, you shouldn't have
any problem. We usually sucrose gradient purify the cell lysates to enrich
for mito, if you ran a different type of gradient I'm sure you could get
good nuclei and/or vacuoles.

Mike C.



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