05 - Homogenization and Extraction - 291

substances have to be separated in an aqueous form (most suitable for lab purposes) as ‘‘extracts’’ by:

  • releasing them from their environment = cell homogenization.

  • purifying them = extraction.

if quantity is favored over quantity in purification/extraction → preparative

  • preparative isolation → isolation of larger quantities (several mg to gms) to be used for further studies.

if quality is favored over quantity in purification/extraction → analytical

  • analytical isolation → isolation of smaller quantities (micrograms or mg) in order to make a precise measurement of certain characteristics.

  • the preparative method is significantly less pure than the analytical.

sequence of steps/protocol of purifying a protein:

  • Develop an assay for the desired protein depending on its characteristics.

    • the protocol differs depending on the characteristic and the part i want to extract specifically.

  • Select the biological source of the protein (cells? Recombinant DNA? Organelle?) considering the following factors:

    • cells

      • source must be considered based on the following factors: quantity - ease of extraction - availability - ease of separation - possibility of storage.

        • types of cells (eg. liver, muscle, blood, plants)

        • location of protein (organelle or out)

          • if it is in an organelle then the organelle must be fractionated to extract the protein.

        • recombinant DNA (very advantageous)

          • allows overexpression of protein.

          • conditions of the protein can be controlled (less contamination and less degradation)

          • possibility of producing mutant proteins.

    • microorganisms and yeasts

      • must be grown in large quantities.

    • plants

      • only a small volume is intracellular.

      • a large amount of plants is required to properly extract and purify proteins.

  • Release the protein from its source and stabilize it in an aqueous buffer system.

  • Fractionate the cell components by physical methods (centrifugation).

  • Separate the biomolecules present by differential solubility.

  • Apply chromatographic procedures.

  • Isoelectric focusing (if desired).

  • Determine purity.