1. LABORATORY 6: PURIFYING THE FLUORESCENT PROTEIN 2014

2. Overview 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

3. PART A: Lyse bacteria cells

4. Reasons 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

5. 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

6. 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

7. PART B: Separate RFP

8. Reasons 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

9. 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

10. 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

11. Separation uses protein folding Unfolded Folded− hydrophobic areas covered

12. Amino 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

13. 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 proteins

14. Protein 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 proteins

15. Protein 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 RFP

16. Results