Goat anti rabbit hrp conjugated secondary antibodies

Embryos were lysed in ice-cold lysis buffer (120 mM NaCl, 50 mM Tris-HCl [pH 8.0], 0.5% NP-40, 1 mM EDTA, and Complete protease inhibitors [Roche]). Lysates were cleared of lipid and yolk by Freon extraction (http://spot.colorado.edu/~klym/) and protein concentration was determined using a BCA Protein Assay Kit (Pierce). Equal amount of protein was resolved on 15% (w/v) polyacrylamide-SDS gels and transferred onto nitrocellulose membranes. To detect Xl-Ink4d protein, we raised a rabbit polyclonal antibody to the C-terminal peptide of Xl-Ink4d1 (amino acid sequence: SQLAAILDPRLASIFELST) and affinity-purified the antibody using the same peptide. This peptide is unique to the predicted Xenopus Xl-Ink4d protein and its sequence is shared between Xl-Ink4d1, Xl-Ink4d2 and the Ink4d of X-Tropical is but not those of Fugu, Mouse or Human. Thus the antibody does not cross react with mouse p19^Ink4d protein (negative data not shown). Membranes were probed with an antibody against α-tubulin (Sigma) as a control for protein loading. Goat anti-rabbit HRP-conjugated secondary antibodies (Amersham) and Super Signal Dura (Pierce) were used to develop the blots.