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.
Zako M, Dong J, Goldberger O, Bernfield M, Gallagher JT, Deakin JA Syndecan-1 and -4 synthesized simultaneously by mouse mammary gland epithelial cells bear heparan sulfate chains that are apparently structurally indistinguishable. J Biol Chem (2003) 10.1074/jbc.M209658200