Principles of Biochemistry (2+1)

Purification of Lysozyme using CM – cellulose

•  Wash the empty column with hot water (90ºC)
•  Fix the column to the stand. Remove the top cap of the column and pack the column with 2.5 ml of CM-cellulose
• Remove the bottom cap and equilibrate the column with 50 ml of 0.1M phosphate buffer
• Break the egg and  separate the egg white.
• To 6 ml of egg white, add an equal volume of distilled water. Mix in the tube provided for 10 minutes to get a homogenous solution
•  Adjust the pH of the egg white to 7.0 by slowly adding neutralizing solution Note: Solution will turn slightly turbid
• Centrifuge the egg white solution at 6000 rpm for 10 minutes. Collect the supernatant
• Save 0.5 ml of the supernatant for measurement of lysozyme activity, label this as crude sample
• Load the remaining portion of the supernatant to equilibrated CM-cellulose column.
•  Replace the top and bottom caps of the column and incubate for 1 hour at room temperature with intermittent mixing
• After an hour, allow the column material to settle. Slowly pipette out or decant the supernatant without disturbing the column. Wash the column with approximately 30 to 40 ml of 1X wash buffer
• Elute lysozyme from the column using 15 ml of 1X elution buffer Start collecting the eluate in test tubes as 2 ml fractions.
• Monitor OD at A280 and pool the fractions that show A280 0.5 and above. Label this as eluate.
• Wash the column with 10 ml of 1M NaCl. Replace the top and bottom caps. Store at 4ºC, for next use.

Note: The packed column can be reused two more times.Discard the column material after 3 uses and pack with fresh 2.5 ml CM-cellulose material.