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Extraction and Purification of Lysozyme from Hen Eggs - Procedure

 

Day 1 - Dialysis and Running Buffer Prep

  1. Obtain a hot plate from the cabinet and bring 200 mL ddH2O to 80oC in a 250 mL beaker.
  2. Cut ~40 cm of dialysis tubing (make sure you wear gloves) and place in the hot water to soak for 30 minutes.
  3. Collect an egg.
  4. Separate the egg white from the yolk as demonstrated by your TA.
  5. Prepare your low salt (50 mM Glycine/50 mM NaCl) buffer to be used for dialysis.
  6. Once the dialysis tubing has rehydrated, pull it out of the bath and rinse well with ddH2O.
  7. Double knot one end of the rubing and open up the other side using forceps. Carefully place the contents of your egg white into the tubing, making sure to leave ~7-8 cm of space for expansion. Double knot the other end of the tube, and make sure both knots are secure.
  8. Using 2 L of your 4 L, place your tubing with egg white into the bucket used to make the low salt buffer, being careful to minimize kinking as much as possible.
  9. Begin to prepare you Sephadex resin and running buffers to be used for separation in the coming days. Prepare these according to the calculations made from your pre-lab. If you do not finish today, you may finish during the interim day, but they must ALL BE READY before the nex lab meeting! Make sure these are all stored in their own containers.

Interim Day

  1. Exchange the buffer in your dialysis tub with a fresh 1 L aliquot of buffer and let your egg white continue to dialyze until the next lab meeting.
  2. Finish preparing your Sephadex resin and running buffers if you have not done so yet.
  3. Set up the supplied chromatography column on a ring stand with a clamp, and fill the column half full with your low salt buffer.
  4. Remove your pre-prepared CM-Sephadex-25 resin from the cold room and give it a quick shake to disturb the resin.On your column, use a wax pencil to denote a 3 cm level from where the filter is. This will be the level you will fill the column up to with the resin.
  5. 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.
  6. 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 low salt buffer through the column. Close stopcock when buffer reaches 1 cm above the bed.
  7. Label and Store your prepared column in the cold room.

Day 2 - Sample Collection through Ion-Exchange Chromatography

  1. Collect your column, buffers and egg white solution from the cold room.
  2. Set up your column for use.
  3. Dilute your egg white (1:4 v/v) using the low salt buffer. Use about 10 mL of your egg white for this.
  4. Homogenize the solution (8 strokes)
  5. Strain the solution into a clean, dry beaker using the shirt fabric provided. Make sure that as little coagulant as possible ends up in your strained sample!
  6. Repeat the straining step an additional 3-4 times.
  7. Obtain 18 clean, dry test tubes for collection.
  8. Remove plastic from column and add 5 mL of low salt buffer to the column.
  9. Open the stopcock and allow ~3 mL of the buffer to drain. Collect the 3 mL in a test tube.
  10. Close the stopcock and load your first 3 mL sample of egg white solution into the column.
  11. Open the stopcock and allow the entire 3 mL sample to elute. Collect this eluent for later analysis.
  12. Close the stopcock and load the second 3 mL sample of egg white solution into the column.
  13. Open the stopcock and allow the entire 3 mL sample to elute. Dispose of this eluent.
  14. Close the stopcock and load the third 3 mL sample of egg white solution into the column.
  15. Open the stopcock and allow the entire 3 mL sample to elute. Dispose of this eluent.
  16. Finally it is time to elute the lysozyme. Run a gradient elution from low to high salt concentration using the 5 buffers you created earlier. Collect the eluant in test tubes in 3-mL fractions by repeating Steps 10-15 above (you will need ~18 test tubes). Label the tubes by sequence of collection and store them along with the remaining buffer and leftover dialyzed egg white in the cold room for the next experiment. Make sure to also collect a 3-mL fraction of post-wash (low salt buffer). AGAIN, MAKE SURE EVERYTHING IS WELL LABELED!!
  17. To confirm, you will complete the lab with the following fractions:
    1. Pre-Wash (1)
    2. Egg White (1)
    3. Salt Buffer Gradients (15)
    4. Post-Wash (1)

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.

For a printable version of the procedure click here