ELISA based quantification of Pax6 expression in the developing Zebrafish embryos

BACKGROUND: Transcription factors are the key regulators of metabolic pathways in the cells, tissues and organ development during embryogenesis. Pax6 is a transcription factor involved in vertebrate eye, brain and central nervous system formation during development. PURPOSE: A reliable and sensitive assay for the spatiotemporal expression, quantification and detection of Pax6 is not available so far in zebrafish as a developmental model, hence the objective of this work is to develop quantitative assay in zebrafish embryos. METHODS: The Pax6 transcription factor was purified by heparin agarose affinity chromatography and DEAE cellulose chromatography techniques from the developing zebrafish embryos. The purity was confirmed by SDS-PAGE and western blotting using Pax6 mouse monoclonal antibody. The standard graph was plotted for Pax6 and the expressions in seventeen developmental stages were quantified by indirect ELISA. RESULTS: The maximum expression of Pax6 was detected at 8 hpf (hours post fertilization) and it was quantified as 179 ng/embryo from the average total protein of 9.5 µg/embryo. The zebrafish Pax6 protein was detected as 48 kDa and confirmed by western blotting. CONCLUSION: This study paves way to quantify the level of expression of proteins or transcription factors during early embryonic and larval development or embryogenesis using zebrafish as model system.