Bio-Rad ReadyPrep Sequential Extraction Kit - Manual (Page 7)

Section 5. Instructions for Use
General Guidelines. The goal of the first step is to lyse the
cells of interest directly in Reagent 1 using a physical lysis
procedure. The method of lysis will vary greatly depending on
cell type. Follow standard procedures for cell growth and tis-
sue harvesting and standard methods for physical cell lysis.
For example, bacterial cultures can be harvested by centrifu-
gation then washed in Reagent 1 to remove excess medium.
The cells can then be suspended in Reagent 1 and lysed by
sonication or by liquid shearing, as in a French press. Animal
tissues can be harvested and then homogenized or sonicated
directly in Reagent 1. Plant tissues can be ground in liquid
nitrogen and the powder suspended in Reagent 1 followed by
homogenization if necessary. Tissue culture cells can be
harvested by centrifugation, washed in a medium-free, isoton-
ic buffer and repelleted. It is important to remove all of the
wash buffer from the pellet prior to the addition of Reagent 1.
Sonication or homogenization can be used to achieve
complete lysis. For best 2-D PAGE results, it is generally
worthwhile to treat the homogenates with nucleases. For this,
add a mixture of DNase I and RNase A to final concentrations
of 20–100 µg/ml and 5–25 µg/ml, respectively. Alternatively,
nonspecific endonuclease at 150 units/ml can be used to
degrade nucleic acids present in the mixture.
Use relative amounts of sample cells or tissue and lysis buffer
estimated to yield about 50 mg/ml of protein solution. The
amount of protein in a cell sample can be reasonably
estimated as its dried weight. For example, sonication of a
5
4006167b.qxd 08/28/2000 6:43 PM Page 5