Protein Protocols Protein Protocols

Fractionated Extraction 145
Table 2
Buffer B
Components Mixture Final concentrationsa KCl 1.491 g 0.2 M
Glycerol 20.000 g 20%
Phosphate bufferb 50.000 mL 0.1 M
Buffer B in 90 mL bidistilled water
aConcentrations in 100 mL of buffer B/CHAPS; see Table 6.
bPhosphate buffer: 33 mL 0.2 M NaH2PO4 solution + 67 mL 0.2 M Na2HPO4 solution; resulting
pH: 7.1.
Table 3
Buffer C
Components Mixture Final concentrations
Tris 0.606 g 50 mM
MgSO4 · 7H2O 0.049 g a
Buffer C in 100 mL bidistilled waterb aIf 1 part (mg) of pellet III is homogenized with 1 part (µL) of buffer C (see Table 6), the
resulting concentration of MgSO4 is 1 mM.
bpH 8.0 (room temperature), adjusted with HCl.
Table 4
Protease Inhibitor 2
Components Stock solutions Mixture
Pepstatin A 9.603 mga in 100 mL ethanol 10 mLc PMSF 1.742 gb in 100 mL ethanol 10 mL
Protease inhibitor 2 20 mL
aConcentration in the homogenate (see Table 6): 1.4 µM.
bConcentration in the homogenate (see Table 6): 1.0 mM.
cThe solution was incubated in a 37°C water bath to dissolve pepstatin A. The solution
should be well-capped.
3. Put the frozen liver pieces into the mortar, and add buffer A, and inhibitor 1A and 2. The
required volumes of each of the solutions are calculated as indicated in Table 6. The precise
amount of each solution is pipeted as a drop onto the spoon that was kept in the N 2-box.
The solution immediately forms an ice bead that can easily be transferred into the mortar.
4. Grind all the frozen components in the mortar to powder. Care should be taken that small
pieces of the material do not jump out of the mortar when starting to break up the hard
frozen material.
5. Transfer the powder into a 2-mL Eppendorf tube using a special spatula (Fig. 1). Forceps
are used to freeze the tube briefly in N 2 and then to hold the tube near to the mortar in the
N 2-box. Care is taken not to leave any powder in the mortar or at the pestle. For collection
of this powder, always use the same type of plastic tube. This contributes to the reproducibility
of the following sonication step. Compress the powder collected in the tube by
knocking the tube against the mortar. The powder can be stored at –70°C or immediately
subjected to sonication.