[35S]sulfate (Perkin-Elmer; 600 nCi in 25 nl/embryo) was injected into the blastocoel of stage 9 embryos, and PGs were purified at stage 22. The PGs were extracted in 4 M guanidine, 50 mM sodium acetate, pH 5.8, 0.1% Triton X-100, 1 mM EDTA, 10 mM N-ethylmaleimide and protease inhibitors. The extraction buffer was exchanged to 6 M urea, pH 5.5, 0.1% Triton X-100, 1 mM EDTA, 0.2 M NaCl and protease inhibitors, and the PGs were anion-exchange purified using a DE52 gel. The purified labeled PGs, desalted with water, were treated with lyases as described above. The samples were subsequently deaminated in nitrous acid at pH 1.5 to degrade heparan sulfate (Shively and Conrad, 1976).
