Heparanase Western Blotting

Contributed by Gordon W. Laurie, School of Medicine, University of Virginia, United States

Heparanase Detection method for Western Blotting

• Generate cell lysates (use ‘Lacritin Affinity Binding of SDC1’ lysis buffer).
• Dialyze lysates overnight against binding buffer (10 mM sodium phosphate, pH 7) and apply to a HiTrap heparin affinity purification (Amersham Biosciences, Piscataway NJ).
• Wash with 10 column volumes of binding buffer, then elute using 5 column volumes of elution buffer (10 mM sodium phosphate, 2 M NaCl, pH 7).
• Determine protein concentration by BCA
• Analyze by 10% SDS-PAGE. Detect HPSE with rabbit polyclonal antibodies directed against human heparanase (kindly provided by Israel Vlodavsky, Rappaport Faculty of Medicine, Haifa, Israel)
• Add HRP-conjugated secondary antibody and ECL.

This method is based, with permission, on an original protocol available here.