Materials required

• Weighing machine

• Blotting paper

• Microfuge tube

• Micropipette

• Pipette

• Pipette pump

• Dialysis tubing membrane

• Forceps

• Wash bottle

• Dialysis clamps

• Magnetic stirrer

• Stir bar

• Visicooler

• Pastuer pipette

• Cryo vial

• -20o C freezer

• Cryo box

• Glass rod

• 5ml test tube

• Centrifuge tube rack

• Gel filtration coloum

• Cuvetttes

• UV spectrophotometer

Reagents required

• Horse radish peroxidase

• Sodium periodate solution

• Ethanol storage solution

• 1mM Sodium acetate buffer

• 0.2M Sodium carbonate buffer

• Sodium borohydride solution

• Borate buffer

• 50% Glycerol

• PBS(Phosphate buffered saline) buffer

• Sepharose CL-6B

Procedure

• Dissolve 4mg of peroxidase in 1mL of water.

• Add 200 µL of freshly prepared 0.1 M sodium periodate, and stir the solution for 20 min at room temperature.

• Dialyze the modified enzyme against 1mM sodium acetate buffer (pH 4.4) overnight at 4oC.

• Adjust the pH of the dialyzed enzyme solution by adding 20 µL of 0.2 M sodium carbonate buffer (pH 9.5) and add 1ml of IgG solution. Stir the mixture for 2 h at room temperature.

• Add 100 µL of freshly prepared Sodium borohydride solution (4 mg/mL), and stir the mixture occasionally over a period of 2 h at 4oC.

• Fractionate the mixture by gel filtration on a column of Sepharose CL-6B in PBS and determine the A280 and A403.

• Pool the fractions in the first peak (both A280 and A403 peaks coincide) and stored at -20 oC with Glycerol added at a final concentration of 50%.