Steps of Protein Purification

Every function in every living organism is determined by the function of its proteins. Purifying a protein allows you to crystallize the pure protein in order to discover different amino acid sequences. In the medical sciences, this is a very valuable process which isolates and identifies what the protein is doing in any biological process. Protein purification is also valuable to the discovery and understanding of evolutionary relationships between organisms.

Things You'll Need

  • Bio-Rad protein Assay Reagent
  • Centrifuge
  • Centrifugation tubes
  • 2 Chromatographic systems
  • 2 Chromatogprahic pumps
  • Fraction Collector
  • Spectrometer
  • Buffer components
  • Filter device
  • SDS-page solutions
  • Refractometer
  • Pippets
  • Fraction collector tubes
  • Pippet tips
  • Concentrators
  • Eppendorf tubes
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Instructions

  1. Obtaining the Protein

    • 1

      Conduct a test based on the unique differences in the protein separate from other proteins. This test is called an "Assay" and it is necessary to use when finding the specific protein.

    • 2

      Separate the different structures of the cell to obtain the structure that has the highest concentration of your particular protein.

    • 3

      Decrease the density of the protein to help narrow your search for the exact protein you are looking for. Proteins all vary in density. Develop a homogenate by tearing the cell membrane by centrifugation. The least dense material, which is furthest from the bottom of the centrifuge tube, is called the "supernatant" and must be obtained and put into the centrifuge again---but at a much faster speed. This, in turn, creates a new supernatant. The repetitive process of putting the supernatant in the centrifuge is called differential centrifugation.

    Purifying the Protein

    • 4

      Add salt to the protein. This identifies different levels of solubility, allowing you to isolate the different subunits and structures.

    • 5

      Use different types of chromatography to further isolate the highest concentration of the protein. (See Resources.)

    • 6

      Use an SDS-PAGE analysis to figure out how accurate your protein purification plan was.

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