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3: Methods of Protein Purification and Characterization

  • Page ID
    165178
    • 3.1: Protein Purification
      A successful protein purification procedure can be nothing short of amazing. Whether you are starting off with a recombinant protein which is produced in E. coli, or trying to isolate a protein from some mammalian tissue, you are typically starting with gram quantities of a complex mixture of protein, nucleic acids, polysaccharide, etc. from which you may have to extract milligram (or microgram!) quantities of desired protein at high purity, and hopefully with high yield.
    • 3.2: Cell Disruption
      There are several ways to break open cells.  Whatever method is employed, the crude lysates obtained contain all of the molecules in the cell, and thus, must be further processed to separate the molecules into smaller subsets, or fractions.
    • 3.3: Fractionation
      Fractionation of samples typically starts with centrifugation. Using a centrifuge, one can remove cell debris, and fractionate organelles, and cytoplasm. For example, nuclei, being relatively large, can be spun down at fairly low speeds. Once nuclei have been sedimented, the remaining solution, or supernatant, can be centrifuged at higher speeds to obtain the smaller organelles, like mitochondria. Each of these fractions will contain a subset of the molecules in the cell.
    • 3.4: Chromatography
    • 3.5: Electrophoresis
      DNA molecules are long and loaded with negative charges, thanks to their phosphate backbones. Electrophoretic methods separate large molecules, such as DNA, RNA, and proteins based on their charge and size. For DNA and RNA, the charge of the nucleic acid is proportional to its size (length). For proteins, which do not have a uniform charge, a clever trick is employed to make them mimic nucleic acids.

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