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Protein Purification - Using Column Chromatography and pGlo

Illuminating protein. Fun!

 

​Purpose: Experience packing a HIC (Hydrophobic Interaction Chromatography) chromatography column and purifying protein using pGlo which is easily visible using a pen UV light. 



Materials:  

Gloves

TE Buffer

Lysozyme (10mg/ml)

Plastic Column

HIC Resin

Microcentrifuge

Vortexer

Tabletop Centrifuge

Equilibration Buffer

Binding Buffer

Elution Buffer

Biorad UV PenLight

Glass Tubes

(3) 1.5mL Minifuge tubes

Minifuge tube rack

5mL Pipette

PIpette Aid





Procedure: 

​

  1. Removed frozen pellet and hand thawed pellet.

  2. Observed and photographed pellet with UV pen light.

  3. Added 250 uL of TE Buffer 

  4. Vortexed for approximately 1 minute on Level 3.  (Watched Instructor's tip of using the tube rack and rubbing the microfuge tube up and down (like a washboard) in lieu of vortexing.)

  5.  Spun tube in minifuge on tabletop for about 20 seconds. 

  6. Added 50 uL of 10 mg/ml of lysozyme and mixed gently. (Observed obvious separation  - whole cell lysate.)

  7. Put tube in the freezer and started timer.

  8. Loaded glass tubes in rack to collect the fractions.

  9. Added 1.5 mls diH2O in plastic column to rinse.

  10. Pressure created using the palm of the hand over the top of the column to help create suction and clear the column faster. During this process the structural integrity of the tube was compromised. Glass shards were disposed of in the glass collection box. 

  11. Added 1ml of slurry (resin) in 20% EtOH and mixed tube with transfer pipette, gently.

  12. Added 4 mls of diH20 to side of plastic column to rinse residual EtOH off of resin. 

  13. Retrieved bacteria at 22:16 on timer & thawed by hand.

  14. Added 2mls of equilibration bufferusing 5ml pipette. Added SLOWLY circling along the sides of the tube to preserve the even resin. 

  15. Pellet tube spun @ 14k RPM (Full Speed) for 10 minutes.

  16. Capped the column to keep it from drying out.

  17. Retrieved the spun tube and extracted the supernatant into a new minifuge tube. Pellet tried to suck up with the supernatant so we got a new tip and extracted the pellet, then with a new tip again, got last of the supernatant.



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