Bio-Rad ReadyPrep™ Protein Extraction Kit (Membrane I) User Manual
Page 9
exercised to prevent heating of the sample. Sonicate
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 M2 into the cell
extract. 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 10 min.
4. Transfer the tube to a 37°C heating block or water
bath and incubate for 30 min. Mix the suspension for
30 sec periodically (3 to 4 times).
5. Centrifuge the tube at maximum speed in a
microcentrifuge (~16,000 x g) for 5 min at room
temperature.
6. Examine the tube carefully. You should notice two
phases. Remove and transfer the top layer, containing
the hydrophilic proteins, to a clean tube.
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