Protein Protocols Protein Protocols

146 Klose
Table 5
Sample Diluent
Components Mixture Final concentrations
Urea 1.08 g (= 0.80 mL) 9.000 M
DTT solutiona 0.10 mL 0.070 M
Servalyt, pH 2.0–4.0b 0.10 mL 2.000%
Bidistilled water 1.00 mL 50.000%
Sample diluent 2.00 mL
a See Subheading 2.2, item 5.
b Serva (D-69115, Heidelberg, Germany).
6. For sonication, a calculated number of glass beads (see Table 6 and Note 6) is given to the
sample powder, and the powder is then thawed and kept in ice. Sonication is performed in
a waterbath. The fill height of the water is critical for the sonication effect and should
always be at the level indicated by the instruction manual of the apparatus. Furthermore,
when dipping the sample tube into the water, it is important to do this at a “sonication
center” visible on the concentric water surface motion and noticeable when holding the
tube with the fingers into this center. We prefer a small sonication apparatus that forms
only one sonication center (see Subheading 2.1.). The water must be kept ice-cold. Sonication
is performed for 10 s. Immediately thereafter, the sample is stirred with a thin wire
for 50 s with the tube still being in the ice water. The tube is then kept in ice for 1 min.
Then the next sonication round is started, until a total of six 2-min rounds has been reached
(see Note 6). After sonication, the glass beads are caught with fine forceps, cleaned as
thoroughly as possible at the inner wall of the vial, and removed. The homogenate sticking
on the wall is collected onto the bottom of the tube by a few seconds of spinning. The
homogenate is then frozen in liquid nitrogen.
7. Detach the frozen homogenate in the tube from the wall by quickly knocking the top of the
tube on the table. Transfer the frozen piece of homogenate into a centrifuge tube (before
this, determine the weight of the tube) and thaw. Centrifuge the homogenate at 50,000
rpm (226,000g max.) for 30 min at 4°C.
8. Completely withdraw the supernatant (I) with a Pasteur pipet and fill into a small test tube
the dead weight of which has been determined before. The centrifuge tube is kept on ice,
and the pipet is put into the tube with the tip at the center of the bottom (the pellet sticks to
the wall if a fixed-angle rotor was used). In this position, remainders of the supernatant of
the bottom of the tube and inside the pipet accumulate in the pipet, and are added to the
test tube. Then the supernatant is frozen in liquid nitrogen and stored at –70°C.
9. Weigh the pellet (I) left in the centrifuge tube on ice, and add buffer A at amounts calculated
as indicated in Table 6. Mix the pellet and buffer by vortexing, collect the homogenate
on the bottom by a short spin, freeze in liquid nitrogen, and store at –70°C or treat
further immediately.
10. Grind the homogenate together with inhibitors 1A and 2 to powder as described above for
the liver pieces. Transfer the homogenate frozen from the centrifuge tube to the mortar
after detaching the frozen homogenate from the wall by knocking onto the bottom of the
tube. Transfer the powder back into the used centrifuge tube, taking care that no powder
remains in the mortar.
11. Thaw the powder, and slowly stir the homogenate for 45 min in the cold room.
12. Centrifuge the homogenate as described in step 7.