(334a) Thermal Stabilization of Tissue Samples Using the Denator Stabilizor T1: The Inactivation of Proteases and Elimination of Proteolytic Fragments On Two-Dimensional Gels

An understanding of cellular processes is facilitated only when methods such as two-dimensional gel electrophoresis (2DGE) are capable of isolating without bias the entire protein constituency of cells such that the analysis can provide a comprehensive representation of the proteome. The regulated expression of proteins as it relates to normal or disease processes, the efficacy of therapeutic regime, or the identification of phenotypes requires that specific proteins are accurately quantified. Therefore, reliable methods of sample preparation become necessary to produce meaningful results. Since proteases regulate many cellular processes including the clearance of down-regulated proteins and the cleavage of precursors to produce functional proteins, it is essential to completely abolish all enzymatic activity to elicit a realistic ?snapshot? of the proteome as it occurs in vivo, both qualitatively and quantitatively. Further, the phosphorylation states of proteins may be altered if enzymes involved in phosphorylation and dephosphorylation are not promptly inactivated. Typically, proteolytic activity is curtailed by rapidly freezing the sample and by the inclusion of protease inhibitors and metal chelators. However, freezing preserves the activity of enzymes which is restored when the samples are thawed. The formation of apocryphal protein spots in 2D gels indicates the inability to completely halt enzyme activity before protein fragmentation can occur. Further, alterations of the proteome caused by in vitro degradation may obscure important information of a sample's biological state and hinder the discovery of biomarkers. An alternative is to use rapid heat inactivation to eliminate enzymatic activity. The Denator Stabilizor T1 has been evaluated and shown to improve results by decreasing proteolytic fragments.