Heparan Sulfate Chain Analysis

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

Analysis of SDC1 HS Chains

  • Generate 50% confluent cell cultures in 150-mm culture dishes.
  • Metabolically label with 50 mCi/ml Na235SO4 (1494 Ci/mmol; PerkinElmer, Boston MA) in DMEM for 48 hours as described by Zako et al. (2003). Use both normal and heparanase-1 depleted cells.
  • After washing three times with PBS, generate cell lysates (use ‘Lacritin Affinity Binding of SDC1’ lysis buffer).
  • Preclear lysates by centrifugation (20,000´g) at 4°C, then by passage of lysate supernatant over beads.
  • Determine protein concentration of supernatant by the BCA assay (Pierce, Rockford IL).
  • Affinity precipitate with FGF2-GST or lacritin-intein beads overnight at 4°C.
  • Digest SDC1 bound to beads using chondroitin ABC lyase (MP Biochemicals, Aurora Ohio) for 3 hours at 37°C.
  • Elute SDC1 from beads with 2 M NaCl and then subject to eliminative cleavage and reduction of HS by adjusting to 100 mM NaOH/1 M NaBH4 for 24 hours at 37°C.
  • Neutralize released HS by drop wise addition of 1M HCl.
  • Subject to Sepharose CL-6B column (1´57cm) gel filtration chromatography in PBS at a flow rate 16 ml/h.
  • Measure radioactivity by liquid scintillation counting.
  • Prior to running the columns, determine the void volume (V0, fraction 26) and total column volume (Vt, fraction 62) using dextran blue and sodium dichromate as markers.