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LABORATORY 6: PURIFYING THE FLUORESCENT PROTEIN. 2014. Overview. In Laboratory 5 , students incorporated recombinant plasmid with rfp gene into a living cell and confirmed that transformation was successful
  • In Laboratory 5, students incorporated recombinant plasmid with rfp gene into a living cell and confirmed that transformation was successful
  • In Laboratory 6, students will obtain cell proteins and purify red fluorescent protein from transformed cells grown in shaker flask overnight
  • PART A: Lyse bacteria cellsReasons for lysis
  • Soluble proteins made by cell, including red fluorescent protein, are dissolved in cell cytoplasm
  • Only way to access soluble proteins is to lyse (break open) cell
  • After lysis, soluble proteins can be easily separated from insoluble structural proteins
  • Procedure
  • Obtain 1mL cell culture in tube
  • Centrifuge for 5 min and identify location of red fluorescent protein
  • Remove supernatant with pipette
  • Add another 1mL of cell culture to tube and centrifuge for 5 min
  • Remove supernatant with pipette
  • Procedure
  • Tip tube and remove last bit of supernatant with pipette, without touching cell pellet
  • Add elution buffer and drag closed tube across tube rack to resuspend cells
  • Add lysis buffer and incubate cells overnight at room temperature to release proteins from the cells
  • PART B: Separate RFPReasons for separation
  • Although the bacteria make a lot of red fluorescent protein, there are up to 1,000 other proteins in a living cell
  • Those other proteins might interfere with intended use of RFP or of any other protein you are isolating
  • Pharmaceutical companies require purified protein
  • Procedure
  • Centrifuge cells (be sure to balance), and remove supernatant with RFP
  • Add binding buffer to the supernatant
  • Add supernatant to column and drain
  • Add wash buffer to column and drain
  • Add elution buffer to column and collect RFP
  • Procedure
  • Always add liquids to the side of column
  • Never touch the resin with pipette
  • Drain until 2 mm of liquid is left above the column and never expose the resin to air
  • Wait until each solution has drained before adding another solution
  • Separation uses protein foldingUnfolded Folded− hydrophobic areas coveredAmino acids’ electric charge
  • Proteins have hydrophobic and hydrophilic amino acids
  • Difference is electric charge distribution
  • Hydrophobic amino acids ARE NOT attracted to water molecules
  • Hydrophilic proteins ARE attracted to water molecules
  • Protein folding in binding buffer
  • In binding buffer, hydrophobic proteins unfold
  • Unfolded hydrophobic proteins adhere to the hydrophobic column resin
  • Folded hydrophilic proteins never adhere to the column
  • Hydrophilic proteinsProtein folding in wash buffer
  • In wash buffer, moderately hydrophobic proteins fold
  • Highly hydrophobic proteins, including RFP, stay unfolded
  • Folded moderately hydrophobic proteins are released from the column
  • Moderately hydrophobic proteinsProtein folding in elution buffer
  • In elution buffer, highly hydrophobic proteins, including RFP, fold
  • Folded highly hydrophobic proteins, including RFP, are released from the column
  • RFP can be collected
  • RFPResults
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