Purification and Characterization of Heparan Sulfate

1 g (wet weight) C57/Bl6 embryonic day 18 mouse embryos or D. melanogaster 1^st–3^rd instar embryos were homogenized and digested overnight in 320 mM NaCl and 100 mM sodium acetate (pH 5.5) containing 1 mg/mL pronase at 40°C. The digested samples were diluted 1:3 in water and 2.5-mL aliquots were applied to DEAE Sephacel columns. HS was eluted and applied to PD-10 (Sephadex G25) columns (GE Healthcare), lyophilized, redissolved in 20 μL water, digested with chondroitinase ABC overnight as indicated, and again purified by DEAE chromatography. Samples were diluted and again applied to PD-10 columns prior to lyophilization. β-elimination of peptides was omitted from this purification protocol to allow for HS coupling to NHS-activated Sepharose via the attached peptides. We confirmed efficient HS coupling to NHS-activated Hi-Trap FPLC columns by using soluble alkaline phosphatase-coupled Fgf8 and VEGF as previously described (Farshi et al., 2011 (link)). HS binding of Shh/Hh was then determined by FPLC (Äkta protein purifier). Samples were applied to the columns in the absence of salt, and bound material was eluted with a linear 0–1 M NaCl gradient in 0.1 M phosphate buffer (pH 7.0). Eluted fractions were quantified as described above. Shh and Hh binding to heparin columns (GE Healthcare) was carried out with the same protocol, except for elution in a linear 0–1.5 M NaCl gradient in 0.1 M sodium phosphate buffer (pH 7.0).