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

Note: If using sonication, care must be exercised to prevent
heating of the sample. The temperature of the sample
should not be allowed to rise above 30°C. Similarly, if the
sample becomes too cold, precipitation of the urea and
thiourea can occur. If this happens, gently warm the sample
until the precipitate dissolves before proceeding further.
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 The addition of
glass beads or use of a Bead Beater (BioSpec Products) can
greatly improve cell lysis of these sample types.
4. Centrifuge the tube at maximum speed in a
microcentrifuge (~16,000 x g) for 20–30 min at
18–20°C to pellet cell debris.
5. Remove and transfer the supernatant to a clean tube
and discard any insoluble pellet.
6. The sample is now ready to be loaded onto IPG strips.
Unused extract should be stored in aliquots at –80°C.
Note when working with plant leaf samples: for best 2-D
separation results treat the sample using the ReadyPrep
2-D Cleanup Kit (Bio-Rad catalog #163-2130) prior to
performing IEF.
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