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Protein Purification Part 2

Tham khảo tài liệu 'protein purification part 2', khoa học tự nhiên, công nghệ sinh học phục vụ nhu cầu học tập, nghiên cứu và làm việc hiệu quả | 8 Protein Purification 75 saturated ammonium sulfate solution containing EDTA to chelate calcium the photocytes were lysed with dilute EDTA solution. A gentle vacuum applied to the suction flask released an amazingly bright stream of fluorescence that was captured in the 4-liter vacuum flask. The extract was precipitated with solid ammonium sulfate the precipitated protein being trapped on a smaller cake of celite or collected by centrifugation. These procedures were developed by Dr. John Blinks Blinks et. al. 1976 . Fig. 3. Underwater photograph of the jellyfish Aequorea victoria. Photograph is courtesy of R. Shimek of the University of Washington s Friday Harbor Laboratories. Soybean peroxidase extraction just requires that the pulverized hulls be stirred in five volumes of distilled water for one hour. Viscosity Reduction and Particle Removal As one might imagine extracts of whole coelenterates or coelenterate tissues jellyfish or sea pansies present a huge problem with viscosity. Aside from water the animals are almost entirely composed of connective tissue and very high molecular weight proteoglycans. For 17 seasons we solved the viscosity problem by passing crude extracts of jellyfish photocytes and surrounding tissues through an 8-liter gel filtration column of P-100 BioGel our next step after ammonium sulfate precipitation . The void volume fraction calibrated to have a molecular weight of 40 million Daltons or greater contained most of the viscosity and none of the GFP. But while this 3-day procedure worked quite well as a viscosity reduction method each gel filtration run could handle one at a time only 5 of a season s collection. Larger amounts of extract invariably fouled the column. If one includes the frequent column washes required to maintain reasonable flow it takes 5-6 months to pass a season s worth of jellyfish extract through the column. It was not without trying many alternative methods that we settled on this highly unusual first .