Optimal standard conditions for protein extraction and solubilization from frozen tissue samples have been examined. Quantitative differences in specific protein amounts or post-translational modifications underlie many, if not all, disease states. Maximal and standardized extraction and solubilization of protein from diseased or healthy tissue is important to make the whole protein complement available for proteomic analysis, and to make the best use of a precious resource. Minimal degradation of the protein amino acid backbone, or of phosphorylated amino acid side chains, during sample preparation is essential to preserve the analytical utility of the extract. We have investigated parameters of brain tissue disintegration, and of extraction/solubilization temperature, time and volume and have reached 98% extraction of brain tissue, corresponding to about 100 microg protein per mg tissue wet weight, by an SDS-based method: Tissue disintegration in the frozen state, by ball mill grinding followed by extraction and solubilization in 2% SDS for 10 min, at 70 degrees C, in a volume corresponding to ten times the tissue wet weight, with shaking. The treatment with SDS sample buffer can inhibit protease and phosphatase activity. Moreover, endogenous enzymes can be inhibited by incubation at high pH. The resulting protein extracts can be used for both one-dimensional SDS gel-electrophoresis and for two-dimensional isoelectric focusing/SDS electrophoresis. The proposed standard protocol has the potential to find wide application where protein extraction, solubilization, identification and quantitation from cryopreserved clinical samples are desirable.