Focusing on lysis for protein extraction, here are some of the reagents available for fast and efficient lysis of some of the most common cell types you might be using. Bugbuster gently breaks open E. The lysis procedure can be enhanced by the addition of benzonase a nuclease and lysozyme. Note that standard bugbuster contains primary amines, but an alternative formulation without primary amines is available. Popculture is a detergent-based reagent that can be added directly to an E. It is mainly used to prepare crude extracts for affinity purification by can also be used for some screening applications.
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Center for Biotechnology Protocols. Extraction of active protein from E. 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. 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. A representative 2D gel of proteins extracted from E.
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.
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