Extraction of active protein from E. coli without sonication BugBuster (primary amine-free) Protein Extraction Reagent. Is a special formulation of BugBuster designed for applications where primary amines would interfere if present in the protein extract, such as protein immobilization or cross-linking. ProteoExtract® Complete Bacterial Proteome Extraction Kit (C-PEK) is designed for fast and easy extraction of total proteins from bacteria without the need for sonication or precipitation
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 interactionsbetween 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 Complete Bacterial Proteome Extraction Kit (C-PEK) that allows sample preparation from both gram-negative and gram-positive bacterial cells intwo steps in a microcentrifuge tube minimizing protein loss. 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 E. coli is shown in figure 1.
C-PEK is designed to extract virtually all proteins from a given bacterial cell. 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 extensively forextraction and 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 be visualized on 2DE-gels. For degradation of nucleic acids, Benzonase, a proprietary protease-free non-specific nuclease,