TAILIEUCHUNG - Protein Purification Part 3

Tham khảo tài liệu 'protein purification part 3', khoa học tự nhiên, công nghệ sinh học phục vụ nhu cầu học tập, nghiên cứu và làm việc hiệu quả | The Art of Protein Purification 23 Fig. 7. Tube showing Stage- I of Three-Phase Partitioning. Three phases are formed after centrifugation. Layer A contains t- butanol B is a thick pancake layer of precipitated material and C is aqueous ammonium sulfate solution containing GFP. Meanwhile membrane phospholipids triacylglycerols pigments dyes cholesterol and other steroids fats oils and miscellaneous lipids become dissolved in the upper layer. Exposure of a complex macromolecular mixture to both t-butanol and the now higher salt concentration causes massive precipitation. The precipitate settles below the organic layer as a thick pancake of congealed protein nucleic acids cell walls and other unwanted materials Fig. 8 . While the t-butanol under the influence of high salt has dissolved the cell membrane it has not affected the cell wall. Normally with membrane dissolved nearly every macromolecule in the cell can escape to the outside through the cell wall. But stressed by the high concentration of ammonium sulfate t-butanol binds to anything that is even slightly hydrophobic causing massive precipitation of most of the proteins and virtually all chromosomal DNA. These aggregates are too large to exit through the cell wall pores so they remain entombed inside the cell behind the cell wall barrier. The binding of t-butanol to these macromolecules whether they have remained within the cell or escaped to the outside lowers the density of the precipitated macromolecules to such a point that the still intact cells with their entombed macromolecules easily float above the ammonium sulfate solution below. The whole cell mass forms a thick rubbery mat that floats above the salt solution Fig. 8 . As centrifugation simply speeds up the formation of three layers the process can be scaled up to almost any volume by simple gravitational settling in a large tank. There is no limit to the scale-up potential. After loss of 20 of the water to the overlying alcohol layer GFP is still .

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