Embryos (n=250 to investigate the activity at different stages; n=50 to investigate the effect of morpholinos) were lysed in 1 ml PBS, 1% Triton X-100, 1 mM DTT, 1 mM EDTA and protease inhibitors (Complete Mini; Roche, 04693124001). Cleared supernatant was exchanged with dialysis buffer (20 mM MES, pH 5.5 at 37°C, 10% glycerol, 0.5 mM EDTA, 0.1% Triton X-100, 1 mM PMSF and 1 mM DTT) using PD10 columns (GE Healthcare). The protein content was measured by performing a Bradford assay (Bio-Rad), using bovine serum albumin (BSA) as the standard. Equal amounts of protein were assayed in a final 100 µl reaction buffer that was composed of dialysis buffer as previously described, which contained 2 mM MnCl2, 0.5% NP-40, 100 µg BSA and 30,000 dpm of the labeled chondroitin substrate ([5-3H]defructosylated, K4 prepared as previously described; Hannesson et al., 1996). Incubations were conducted for 20 h at 37°C, and the released tritium was quantified as previously described (Pacheco et al., 2009a). During the assay, it was determined that the inhibitory components in the lysates varied in the embryos of
