Heparin affinity chromatography

Protein samples produced in HEK293T cells were freshly purified by SEC and then adjusted in 50 mM Tris, pH 7.5, 0.25 M NaCl. Purified protein (0.5 mg) was loaded onto a 1 ml HiTrap heparin HP column (GE Healthcare Life Sciences) equilibrated in 20 mM Tris, pH 7.5, 0.25 M NaCl and eluted with a linear NaCl gradient to 20 mM Tris, pH 7.5, 2 M NaCl, 5% (wt/vol) glycerol over 10 column volumes. Notably, we found that Norrin–Fz4_CRD complex tends to partially disassemble (Fz4_CRD detected in flow-through; Figure 7A) during sample preparation for the heparin binding assay (NaCl concentration was reduced from 0.5M to 0.25M).