Bio-Rad ReadyPrep Sequential Extraction Kit - Manual (Page 8)

suspension of 0.5 g (wet weight — equivalent to about 100
mg dry weight) of Escherichia coli in 2 ml of Reagent 1 results
in effective lysis and fractionation.
Lysis conditions are very important to the success of a
sequential extraction. Proper conditions for thorough lysis
should be determined empirically. For example, overly
aggressive sonication can denature some proteins, while
insufficient sonication can leave some of the cells intact.
Several texts and Internet sites can be consulted for details of
cell culturing and growth and for methods to lyse a wide
variety of cell types. For example, the ExPASy
(http://www.expasy.ch) and Biobase (http://biobase.dk/cgi-
bin/celis) web sites with their associated links contain much
information on sample preparation. Several texts, such as
References 6–9, are valuable sources of information.
Sequential Extraction. Refer to the schematic illustration
for a flow chart of the sequential extraction procedure.
Extraction 1
1 Place the desired starting mass of cells or tissue in a suitable
tube and add Reagent 1 in a ratio estimated to yield at least
50 mg/ml of protein upon lysis.
2 Lyse the cells by sonication or any other suitable physical
means.
3 Transfer the suspension to centrifuge tubes and centrifuge
the sample until firm pellets form. For example, centrifuge E.
coli
lysates at top speed in a benchtop microcentrifuge at
room temperature for 10 min.
4 Recover the supernatant and determine its protein content
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