242 - Validating Zebrafish Chaf1b Antibody

Zebrafish embryos that are homozygous for gef mutant alleles experience a small-eye phenotype, due to a coding change in the chaf1b gene that results in a prematurely truncated protein. It's thought that the Chaf1b protein is required for all dividing cells to survive the DNA replication phase of the cell cycle. Contradicting these findings, the proliferating cells of the gef mutant embryos do not start to prematurely die until they are two days old. One question of delayed death in zebrafish is whether the normal protein can be provided by the heterozygous mother. If so, this would account for how the dividing cells are surviving for days in the developing mutant embryo. This study will test for Chaf1b protein in cells dying during the proliferation step in an attempt to detect Chaf1b protein. It is hypothesized that surviving, not dying, retinal cells may have the normal Chaf1b protein which could be detected by anti-Chaf1b antibodies. This research will verify this question by using antibodies against Chaf1b. Previous research obtained unclear Western Blot results that suggested 3 dpf gef mutant embryos lack detectable Chaf1b protein. Thus, validation of Chaf1b antibodies will be analyzed with a Dot Blot to confirm the efficiency of the antibody used in the Western Blot. If the antibody is valid, it is hypothesized that Dot Blot results will display wild-type zebrafish embryo extract glowing under chemiluminescence through anti-Chaf1b immunoprecipitation, while there will be reduced levels of Chaf1b detected in 3 dpf gef mutant embryos compared to wild-type.