NOVAGEN POP culture Reagent

Novagen

PopCulture Reagent* is a buffered mixture of concentrated detergents formulated to extractproteins 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 protectsprotein from the pH extremes produced in high density culture media. Recombinant proteinscan be assayed directly or purified by adding an aff inity matrix, washing the matrix:targetprotein complex to remove culture medium and cellular contaminants and eluting the purifiedprotein from the matrix. Purification of fusion proteins from total culture extracts has beendemonstrated using both IMAC and GST affinity chromatography methods (Grabski et al., 2001).To further enhance the PopCulture purification procedure, lysozyme and/or Benzonase®Nuclease may be added. Lysozyme cleaves a peptidoglycan bond in the E. coli cell wall,enhancing cell lysis and increasing the yield of protein (Inouye et al., 1973; Grabski et al., 2001).Proteins may be expressed in a host encoding T7 lysozyme (pLysS host) or exogenous lysozymemay be added after the PopCulture Reagent. Benzonase Nuclease may also be added to degradeendogenous nucleic acids that may interfere with purification due to high viscosity.The PopCulture protein purification procedure is ideally suited to high throughput (HT) roboticprocessing of samples for proteomics research or any application that would benefit from theincreased speed and convenience. The magnetic agarose capture resins (e.g. GST•Bind™Magnetic Agarose Beads, His•Bind® Magnetic Agarose Beads) are optimal for HT applicationssince the entire procedure can be performed in a single tube without the need for columns orcentrifugation

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