Isolation of Lysozyme

on of Isolation and Characterization of Lysozyme using Ion-Exchange Chromatography, High Performance Liquid Chromatography, and Capillary Electrophoresis

Eamonn F. Healy, Department of Chemistry, St. Edward’s University, Austin, TX 78704. Experimental Preparation of Buffer Solutions a) 100 mls of Phosphate buffer (0.1 mol L-1 / pH=8.0 / 0.2 M Na+) - this is the start buffer: (i) (ii) (iii) prepare 1L of 0.2 M NaOH(aq) prepare 1L of 0.2 M sodium dihydrogen phosphate(aq) add 47 mls of (i) to 50 mls of (ii) and dilute to 100 mls to obtain the desired pH of 8.0. b) To 100 ml volumes of this phosphate buffer solution add 1.169g , 2.338g and 3.507g respectively of NaCl to generate phosphate buffer solutions of ionic strength 0.4M, 0.6M, and 0.8M respectively - these are the elution buffers. c) To 100 ml volumes of this phosphate buffer solution add 4.676 of NaCl to generate a phosphate buffer solution of ionic strength1.0 M - this is the regeneration buffer.

Isolation of Lysozyme using Ion-Exchage Procedure: Extraction of crude protein

Crack and egg and discard the yolk. Filter the egg white through cheesecloth into a small beaker. Dilute the egg aprroximately 3:1 with phosphate buffer (pH 8). Centrifuge at the highest for 15 mins and store the supernatant in refrigerator overnight. Dilute this solution 1:1 with buffer and centrifuge again for 15 mins.The lysozyme is

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in the supernatant so pipette off the top layer.

Procedure: Isolation using Ion Exchange

Remove the twist-off end of the 1 ml column and use either a 1 or 5 ml syringe, with adapter, for pumping. Prepare the column as follows : 1. 2. 3. Wash with 5 mls of start buffer Wash with 5 mls of regeneration buffer Wash with 5 mls of start buffer

Apply 5 mls of crude extract to the column. Wash with 5 mls of start buffer, collect this sample in a vial labelled wash Elute the column with 5 mls in succession of 0.4M, 0.6M, 0.8M and 1M NaCl(aq) collecting each elution in a vial with appropriate...