A semiautomated system for the production and analysis of sucrose density gradients |
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Authors: | C S Lange D F Liberman |
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Institution: | Departments of Radiology (Experimental Radiology), Radiation Biology and Biophysics, and Microbiology, The University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 USA |
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Abstract: | A semiautomated system permitting considerable accuracy, speed and reproducibility in the making and fractionation of sucrose density gradients is described. The system consists of a modified Beckman gradient forming device which makes six gradients simultaneously and delivers them into six 12.5 ml polyallomer centrifuge tubes in such a manner that new material is continuously added to the meniscus of the gradient. The gradients are fractionated three at a time and up to 100 fractions per gradient can be collected automatically directly into scintillation vials with a choice of drop counting or time mode with rinse and automatic addition of scintillation fluid to each vial. The system can process up to six gradients per hour but centrifugation time is usually the limiting factor. With neutral sucrose gradients, sharp, reproducible, monodisperse peaks containing up to 100% of the gradient radioactivity are usually obtained but a smaller monodisperse peak containing as little as 3.5% of the gradient radioactivity can be detected under conditions where some pairs of molecules might tangle or dimerize. The resolution and reproducibility of this system when used with neutral sucrose gradients is at least the equal if not superior to that commonly claimed for alkaline sucrose gradients. |
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