Sample preparation

Tissue samples were frozen immediately after sampling in the operating room, stored in dry ice and kept at -70 C until analysis. Samples of 20 mg were washed in 400 µl buffer (50 mM Tris/HCl pH 7.1, 100 mM KCl, 6 mM EDTA, 5.8 µM benzamidine, 2.1 µM leupeptin) for 30 s, pulverized under liquid nitrogen and solubilized in 100 µl buffer containing 9 M urea, 25 mM Tris/HCl pH 7.1, 50 mM KCl, 3 mM EDTA, 2.9 mM benzamidine, 70 mM dithiothreitol and 2% (v/v) carrier ampholyte (Servalyte, pH 2-4, Serva, Heidelberg). Finally, 2 µl of additional protease inhibitors (5 µM pepstatin, 50 mM phenylmethylsulfonyl fluoride in ethanol) were added. The resulting protein concentration was about 20 µg/µl. 150 µg protein were loaded on each gel. The protein concentration was determined according to Lowry [9], modified according to Petersen [10] , and serum albumin was used as a standard.

Two-dimensional gel electrophoresis

The isoelectric focusing for the first dimension and the sodium dodecyl sulphate polyacrylamide gel electrophoresis for the second dimension were used as described previously [11]. The isoelectric focusing gels contained 9 M urea, 4% (w/v) acrylamide, 5% (v/v) glycerol, 0.3% (w/v) piperazine diacrylamide, 0.06% (v/v) N,N,N',N'-tetramethylethylendiamine and a complex of 4% (v/v) ampholyte mixture pH 2-11. The gels were 20.2 cm long and 0.15 cm thick. Samples were loaded at the anodic site. Focusing was done for 21 h for a total of 8867 Vh. After focusing the gels were equilibrated for 10 min with buffer ( 58.8 mM Tris/phosphate pH 6.9, 40% (v/v) glycerol, 65 mM dithiothreitol, 3% (w/v) sodium dodecyl sulphate ) and frozen at - 70 C.

For the second dimension, SDS-PAGE according to Laemmli [12] was used omitting the stacking gel. Gels contained 15.0% (w/v) acrylamide and 0.2% (w/v) bisacrylamide, 0.05% (v/v) N,N,N',N'-tetramethylethylendiamin, 0.1% (w/v) SDS, 90 mM Tris/HCl and 285 mM Tris-base. The gels measured 23 x 29 x 0.15 cm. Gels were run for 7.5 hours. After completion of SDS-PAGE the gels were fixed overnight in fixation solution consisting of ethanol / water / acetic acid (50 / 40 / 10, by vol.). For the detection of proteins silver staining was used [13, 14]. After staining the gels were dried at 70 C under vacuum.

REFERENCES

[9] Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J., J. Biol. Chem. 1951, 193, 265-275

[10] Peterson, G. L., Anal. Biochem., 1977, 83, 346-356.

[11] Klose, J., in: Tschesche, H. (Ed.), Modern Methods in Protein Chemistry - Review Articles. Walter de Gruyter Verlag, Berlin, New York 1983, pp. 49-78.

[12] Laemmli, U. K., Nature, 1970, 227, 680-685.

[13] Heukeshoven, J., Dernick, R., Electrophoresis, 1985, 6, 103-112

[14] Jungblut, P., Seifert, R., Biochem.Biophys.Methods , 1990, 21, 47-58

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