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 different developmental stages. To remove these inhibitions and to increase the sensitivity of the assay, in particular during the early stages, the lysates were enriched in epimerase activity by purification on Red-Sepharose gel (Pacheco et al., 2009a; Gustafsson et al., 2014). A total of 2 mg of lysates in 1 ml 60 mM MES pH 6.5 and 100 mM NaCl were prepared and batch-incubated overnight at 4°C with 30 µl Red-Sepharose gel (GE Healthcare). The gel was washed with incubation buffer, and the epimerases were eluted with 200 µl dialysis buffer, 2 M NaCl and 1 mg/ml BSA as the carrier. The samples were desalted on membranes by using dialysis buffer, and 50 µl of the preparation was subsequently assayed.
