Affinity Purification of HRP

1 M a-methyl-D-glucoside or -mannoside in PBS C 1 M NaCl, 1 mM CaCl2,1 mM mgCl2,0.02% Thimerosal (w/v) in 0.1 M acetate buffer, pH 6.0, supplemented with 1 mM MnCl2 after adjusting pH 

Wash a column with 5 ml Concanavalin A bed (1 x 6.4 cm) with 50 ml of Soln. A at RT. Apply up to 20 ml of the above HRP solution. When all of the dark brown solution is within the gel, wash with Soln. A with a flow rate of about 1 ml/min at RT, until the eluate is colorless. If the capacity of the column is not sufficient, the eluate may be rechromatographed after regeneration of the column. Elute the bound colored HRP with Soln. B at 1 ml/min. Regenerate the column with 25 ml of Soln. C and store it in a refrigerator until reusing. 

5 Affinity Chromatography of Immunoglobulins on immobiiized Antibodies (Immunoaffinity Chromatography, iAC) 

5 M phosphate buffer, pH 7.5, or 0.5 M Tris buffer pH 8.0 BrCN-immobilized IgG 

Dilute the respective antiserum, e.g., goat anti-(rabbit IgG) antiserum, to about 10 mg/ml with Soln. A and centrifuge or filter through a 0.45-pm membrane filter. 

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