Two-dimensional-electrophoresis (2DE) remains the highest resolution technique for protein separation when complex samples need to be arrayed for characterization, as in proteomics. Pretreatment of samples for isoelectric focusing (IEF) followed by polyacrylamide gel electrophoresis (SDS-PAGE) involves solubilization, denaturation and reduction to completely break any interactions between the proteins and to remove non-protein sample components. Ideally, to avoid protein losses, one would achieve complete sample solubilization in a single step and thus eliminate unnecessary handling. The prerequisite of a successful proteome analysis is a standardized and reproducible sample preparation procedure for the biological sample of choice. Having this in mind, we have developed the Mammalian Complete ProteoExtract Kit (C-PEK) that allows sample preparation from both tissue culture cells and tissues in two steps in a microcentrifuge tube minimizing protein loss. Extraction from tissue culture cells and tissues with C-PEK results in a total protein sample solubilized in the presence of ultrapure chemicals that is ready for two-dimensional separation with high reproducibility. The complete cell extract is ideally suited for overview gels and for fast screening of the influence of experimental parameters on protein expression or modification by means of e.g. 2DE-Western-Blotting or autoradiography of cell extracts. A representative 2D gel of proteins extracted from a tissue culture cell line is shown in figure 1.
C-PEK is designed to extract virtually all proteins from a given tissue culture cell or mammalian tissue. It includes a hypotonic buffer for cell resuspension and lysis and a denaturing extraction reagent for the solubilization of proteins. The Extraction Reagent contains compounds that have previously been used for extraction as also new reagents with increasing solubilization power that improve
solubilization of proteins prior to 2DE resulting in an increased total number of spots that can to be visualized on 2DE-gels. For degradation of nucleic acids, Benzonase, a proprietary protease-free non-specific nuclease, is included, which leads to a clarified, non-viscous protein solution showing higher resolution in 2DE-gels. Reduction of extracted proteins is performed with DTT. Despite that DTT is negatively charged at alkaline pH, it was observed to be superior over TBP in the complete proteome extraction procedure. Furthermore, besides being difficult to handle due to spontaneous decomposition, TBP was found to be unstable in concentrated urea solutions as used in sample preparation for 2DE . The addition of a protease inhibitor cocktail during cell extraction was found to be unnecessary as the overall protease activity was sufficiently inhibited by the Extraction Reagent in tested cases. For cell lysis and protein extraction, cells or tissues are frozen and thawed by resuspension in the Resuspension Buffer. Subsequently, proteins are extracted with the Extraction Reagent. The lysis conditions are very important for the success of the extraction procedure and strongly depend on the cell type. While tissue culture cells are satisfactorily disrupted by the combination of freezing and thawing in a hypotonic buffer, tissues require a more vigorous disruption using a bead mill for efficient disruption and homogenization. In order to minimize laborious optimization work, special extraction procedures for tissue culture cells and for tissues have been developed using the Mammalian Complete ProteoExtract Kit and are described below. Use the information given in table 1 as a guideline to score for the success of your extraction procedure.