Protein Protocols Protein Protocols

156 Klose
tion. The calculation of the control values is indicated in Table 6. In the following, an
example is given from a real experiment. From a series of 73 individual mouse hearts, the
SI + II fractions were prepared and the control values B ÷ A (see Table 6) were calculated:
60 samples showed values between 1.97 and 2.10, three samples between 1.94 and
1.96, and six samples between 2.11 and 2.13. Four samples with greater deviations (1.90,
1.91, 2.16, 2.24) were excluded from the investigation. The range 1.97 and 2.10 (mean
2.04 ± 0.04) was taken as standard control value for the preparation of mouse heart SI + II
samples.
5. Amount of tissue used as starting material: The amount of tissues given in Table 6 can be
increased, but should not exceed 500 mg. However, the tissue amounts indicated give
enough sample solutions to run many 2-DE gels, so that less rather than more material can
be used.
6. Sonication: The conditions for sonicating mouse tissue homogenates (liver, brain, heart)
were determined with the aim of breaking the membranes of all the cells and cell nuclei of
the tissue. Three parameters were varied in the experiments: the length of the period of
sonication, the number of repeats of sonication, and the number of glass beads added per
volume of the homogenate. The effect of the various conditions was checked under the
microscope by inspection of the sonicated material. During 10 s of sonication, the temperature
of the homogenate increased from 0°C to 11–12°C. Therefore, sonication was
not performed for more than 10 s. Glass beads are essential for breaking the cellular structures
(membranes).
Since most of the homogenates are rather thick fluids, the beads cannot flow freely.
Consequently, for a given volume of homogenate, a certain number of glass beads is necessary
to expose the homogenate evenly to the sonication effect. This number was standardized
by using the factor 0.034 in calculating the number of glass beads for a given
volume of homogenate.
It follows from the above-mentioned three experimental parameters that the only
parameter that can be varied to manipulate the effect of sonication was the number of
repeats of the 10-s sonication period. Under the conditions described in Subheading 3., the
membranes of all cells were broken and no longer visible under the microscope. However,
a certain number of intact nuclei were still detectable. We did not try to break even
these nuclei by extending sonication. Sonication by using a metal tip cannot be recommended.
We observed heavily disturbed 2-DE patterns as a result of employing this technique:
many protein spots disappeared depending on the extent of sonication, and new
spot series occurred in the upper part of the gel, apparently as a result of aggregation of
protein fragments.
7. Amount of protein applied per gel: The protein samples applied to the IEF gels (see Table 6),
contain about 100 µg protein. There is, however, no need to determine the protein concentration
of each sample prepared in order to obtain protein patterns of reproducible intensities.
The concept of the procedure described here for extracting tissues was to keep the
volume of the extracts in strong correlation to the amount of the starting material (tissue or
pellet) that was extracted. Therefore, by working precisely, the final sample should always
contain nearly the same protein concentration. Accordingly, the reproducibility of the
pattern intensity depends on the precise sample volume applied to the gel—and, of course,
on the protein staining procedure.
The sample volumes per gel given in Table 6 are adapted for silver-staining protocols.
A general guideline may be: decreasing the protein amount per gel and increasing the
staining period is better than the other way around; diluted samples at a reasonable vol-