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Beschreibung

PopCulture Reagent* is a buffered mixture of concentrated detergents formulated to extract proteins from E. coli cells directly in their culture medium. Using this method, cell culture, protein extraction and purification performed in the original culture tube or multiwell plate. PopCulture perforates the E. coli cell wall without denaturing soluble protein and protects protein from the pH extremes produced in high density culture media. Recombinant proteins can be assayed directly or purified by adding an affinity matrix, washing the matrix:target protein complex to remove culture medium and cellular contaminants and eluting the purified protein from the matrix. Purification of fusion proteins from total culture extracts has been demonstrated using both IMAC and GST affinity chromatography methods (1).To further enhance the PopCulture purification procedure, lysozyme and/or Benzonase(R) Nuclease may be added. Lysozyme cleaves a peptidoglycan bond in the E. coli cell wall, enhancing cell lysis and increasing the yield of protein (1, 2). Proteins may be expressed in a host encoding T7 lysozyme (pLysS host) or exogenous lysozyme may be added after the PopCulture Reagent. Benzonase Nuclease may also be added to degrade endogenous nucleic acids that may interfere with purification due to high viscosity.The PopCulture protein purification procedure is ideally suited to high throughput (HT) robotic processing of samples for proteomics research or any application that would benefit from the increased speed and convenience. The magnetic agarose capture resins (e.g. GST&bull,Bind Magnetic Agarose Beads, His&bull,Bind Magnetic Agarose Beads) are optimal for HT applications since the entire procedure can be performed in a single tube without the need for columns or centrifugation.*patent pending