Protein Protocols Protein Protocols

142 Klose
remained is extracted with urea and CHAPS, and the homogenate is centrifuged. The
supernatant is the PE fraction and gives the second protein sample. The final pellet (III)
is suspended into buffer containing benzonase, a DNA-digesting enzyme. The PS (fraction)
is the third protein sample. It is applied to 2-DE without further centrifugation.
Care is taken during the whole procedure to avoid any loss of material. In spite of
that, some material may become lost, for example, by the transfer of the pulverized,
frozen tissue from the mortar to the tube or by removing the glass beads from the
sonicated homogenate. However, this does not lead to a preferential loss of certain
protein species or protein classes.
It is evident that the best conditions for keeping proteins stable and soluble are given
in the living cells (2,3). Therefore, a principle of our tissue extraction procedure was to
extract the so-called soluble proteins (SI + II) as far as possible under natural conditions.
That means keeping the ionic strength of the tissue homogenate at 150–200 mM, the
pH in the range of 7.0–7.5, the protein concentration high, and protecting the proteins
against water by adding glycerol to the buffer. Generally, the best conditions for the
first tissue extraction would be given if the addition of any diluent that disturbs the
natural concentration and milieu of the cell proteins were avoided. We prepared a pure cell
sap from a tissue by homogenizing the tissue without additives (except for protease
inhibitor solutions added in small volumes) followed by high-speed centrifugation, and
extracted the pellet that resulted successively in increasing amounts (0.5, 1, or 2 parts)
of buffer. The series of protein samples obtained were separated by 2-DE and the patterns
compared. The results showed that by increasing the dilution of the proteins, the
number of spots and their intensities decreased in the lower part of the 2-DE patterns
and increased in the upper part. The same phenomenon, but less pronounced, was
observed even when the cell sap already extracted, was diluted successively. Apparently,
low-mol-wt proteins are best dissolved in the pure cell sap and, presumably, tend
to precipitate in more diluted extracts. High-mol-wt proteins, in contrast, become better
dissolved in more diluted samples. This effect was most pronounced in protein
patterns from the liver and not obvious in patterns from heart muscle. This is probably
because of the high protein concentration in the liver cell sap that cannot be reached in
extracts of other organs.
The dependency of the protein solubility on the molecular weight of the proteins is
obscured in 2-DE patterns by another effect that leads to a similar phenomenon. The
higher the concentration of the first tissue extract, the higher the activity of the proteases
released by breaking the cellular structure by homogenization. Protein patterns
from pure liver cell sap, extracted without protease inhibitors (but even with inhibitors)
showed an enormous number of spots in the lower part of the gel and a rather depleted
pattern in the upper part. In the pH range around 6.0, the protein spots disappeared
almost completely, in the upper as well as in the lower part, suggesting that these proteins
are most sensitive to the proteases. By extracting the tissue or first pellet with
increasing amounts of buffer, the 2-DE pattern (spot number and intensity) shifted
from the lower part to the upper part of the gel. Again, this observation was made
particularly in liver.
The consequence of these observations for our protein extraction procedure was to
gain the supernatant I and II at concentrations that keep all the soluble proteins in
solution, but do not reach a level where proteases cannot be inhibited effectively
enough. We introduced buffer factors that determine the concentration of the different