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Extraction and Purification of Lysozyme from Hen Eggs - Procedure

 

Day 1

  1. Collect an egg.
  2. Separate the egg white from the yolk as demonstrated by your TA.
  3. Prepare your low salt (50 mM glycine/50 mM NaCl) buffer.
  4. Dilute your egg white (5:1 v/v) using the low salt buffer.
  5. Homogenize the solution (8 strokes)
  6. Strain the solution into a clean, dry beaker using 4 layers of cheese cloth.
  7. Filter (gravity) the solution through a p5 filter paper
  8. While the solution is filtering, prepare the CM-Sephadex-25 resin according to the procedure you produced from the pre-lab question. Place it in a 100-mL beaker covering the top with parafilm and storing it for use on the second day of lab this week.
  9. Collect the filtered solution, and prepare it for storage. Label and store the solution in the cold room as instructed by your TA.
  10. Prepare the remaining buffers you will need for your separation based on the calculations you performed in your pre-lab.
  11. Before leaving make sure that you have prepped and properly stored your remaining low sat buffer, separation running buffers, Sephadex resin, and any leftover egg white. Each should be well labeled and stored in the walk-in refrigerator within the lab. Please make sure everything is WELL LABELED!!
 

Interim Day

  1. Set up the supplied chromatography column on a ring stand with a clamp, and fill the column half full with your low salt buffer.
  2. Remove your pre-prepared CM-Sephadex-25 resin from the cold room and give it a quick shake to disturb the resin.
  3. After opening the stopcock of the column allowing the buffer to drain through, quickly pour your resin into the column. Continue to drain the buffer through the column until a small amount of buffer sits above the packed resin. (1-cm above should be appropriate) Calculate the actual bed volume and record.
  4. So as not to disturb the sephadex bed, pour 10 mL buffer into the column so that it runs down the sides of the column. Open the stopcock to run the wash buffer through the column, collect and keep a 3mL column wash eluant in a test tube. Close stopcock when buffer reaches 1cm above the bed.
  5. Label and Store your prepared column in the cold room.

Day 2

  1. Collect your column, buffers and egg white solution from the cold room.
  2. Set up your column for use.
  3. Obtain 17clean, dry test tubes for collection.
  4. Remove plastic from column and add 5 mL of running buffer to the column.
  5. Open the stopcock and allow ~3 mL of the buffer to drain. Collect the 3 mL in a test tube.
  6. Close the stopcock and load the first of 3 3mL samples of your egg white solution into the column.
  7. Open the stopcock and allow the entire 3mL sample to elute. Collect this eluent for later analysis.
  8. Close the stopcock and load thesecond of 3 3mL samples of your egg white solution into the column.
  9. Open the stopcock and allow the entire 3mL sample to elute. Dispose of this eluent.
  10. Close the stopcock and load the final of 3 3mL samples of your egg white solution into the column.
  11. Open the stopcock and allow the entire 3mL sample to elute. Dispose of this eluent.
  12. Finally it is time to elute the lysozyme. Run a gradient elution from low to high salt concentration using the 4 dilutions and high salt buffer you created earlier. Collect the eluant in test tubes in 3-mL fractions (you will need ~15 test tubes). Label the tubes by sequence of collection and store them along with the remaining buffer, leftover dialyzed egg white, and the undiluted supernatant from the centrifugation step in the cold room for the next experiment. AGAIN, MAKE SURE EVERYTHING IS WELL LABELED!!

NOTES:
DO NOT LET THE COLUMN RUN DRY.

TRY NOT TO TURN THE TAP OFF, KEEP THE COLUMN RUNNING AND ADD THE NEXT BUFFER WHEN THE PREVIOUS ONE RUNS DOWN TO ABOUT 1cm ABOVE THE BED.

IF YOUR COLUMN RUNS SLOWELY YOU MAY ATTACH A 3mL SYRINGE AND PULL THE SAMPLE OUT OF THE COLUMN. USE THIS ONLY AS A LAST RESORT.

 

Printable PDF version of procedure available here