Protein Protocols Protein Protocols

Fractionated Extraction 143
Fig. 1. Special equipment for the pulverization of frozen tissue (A) Glass mortar and plastic
pestle. (B) Spatula used to transfer the frozen tissue from the mortar to the test tube. A regular
spatula was formed to a small shovel.
Table 1
Buffer A
Components Mixture Final concentrations
Tris 0.606 g 50 mM
KCl 0.746 g 100 mM
Glycerol 20.000 g 20%
Buffer A in 100 mL bidistilled watera a pH 7.1 (room temperature), adjusted with HCl.
extracts of each organ. The optimum concentrations were determined experimentally.
The optimum was considered to be reached when a maximum of spots occurred in the
upper as well as in the lower part of the 2-DE protein pattern. The region around pH 6.0
should not tend to become depleted, starting from the top.
The method described in the following was developed with mouse tissues, but was
found to be applicable in the same manner for the corresponding human tissues.
2. Materials
2.1. Equipment
1. Sonicator for performing sonication in a water bath: A small apparatus is preferred
(Transsonic 310 from Faust, D-78224 Singen, Germany).
2. Glass beads added to the tissue sample for sonication: The size (diameter) of the glass
beads should be 2.0–2.5 mm. The factor 0.034 is calculated for this size of beads (see
Note 6).
3. Mortar and pestle: Form and size of this equipment are shown in Fig. 1. Mortar and pestle
are manufactured from achat or from glass (Spec. LAB, Breiter Weg 33, 12487 Berlin,
Germany). Glass was found to be more stable in liquid nitrogen.