Bio-Rad ReadyPrep Protein Extraction Kit (Signal) - Manual (Page 9)

the sample using 30–40 sec bursts until lysis is
complete (typically 3–4 times). Chill the suspension on
ice for 1 min between each ultrasonic treatment.
Note: Disruption of cells by sonication is dependent on the
cell type. For example, E. coli requires longer sonication
times than animal cells and tissues. Yeast cell disruption
requires even more vigorous sonication, and the addition of
glass beads or use of a Bead Beater (BioSpec Products) can
greatly improve cell lysis.
3. Add an equal volume of chilled buffer S2 into the cell
extract.
4. Vortex the suspension 4–5 times, 60 sec each.
Maintain the tube in an ice-water bath for 30–60 sec
between each vortexing step. At the end of the
vortexing procedure, incubate the tube in an ice-water
bath for 15 min.
5. Centrifuge the tube at maximum speed in a
microcentrifuge (~16,000 x g) for 20 min at 4°C.
Remove and discard the supernatant.
6. Keeping the tube cold at all times, suspend the pellet
in one-third the volume used initially (see step 1) of
chilled buffer S1. Vortex to resuspend. Add an equal
volume of chilled buffer S2 (i.e. one-third the original
volume used in step 3). Repeat steps 4 and 5.
6
4110149A5.qxd 1/15/2003 2:49 PM Page 9