PUBLICATION

Detection and measurement of membrane-bound protein tyrosine kinases in the zebrafish egg

Authors
Wu, W., and Kinsey, W.H.
ID
ZDB-PUB-040329-2
Date
2004
Source
In Germ Cell Protocols, Sperm and Oocyte Analysis. Schatten, H., Ed. Methods Mol. Biol. series. Columbia, MO: University of Missouri   1: 273-284 (Chapter)
Registered Authors
Kinsey, William H., Wu, Wen-Jun
Keywords
none
MeSH Terms
  • Animals
  • Cell Membrane/enzymology*
  • Fertilization in Vitro/methods
  • Ovum/enzymology*
  • Peptides/physiology
  • Phosphorylation
  • Protein Tyrosine Phosphatases/analysis
  • Protein-Tyrosine Kinases/analysis*
  • Proto-Oncogene Proteins/analysis
  • Proto-Oncogene Proteins c-fyn
  • Zebrafish
  • Zebrafish Proteins
PubMed
15037802 Full text @ In Germ Cell Protocols, Sperm and Oocyte Analysis. Schatten, H., Ed. Methods Mol. Biol. series. Columbia, MO: University of Missouri
Abstract
The study of the biochemical events involved in fertilization and egg activation has historically been directed toward marine invertebrate and amphibian eggs, although recent progress has been made in the analysis of a few enzymes that are abundant in mammalian eggs. The zebrafish system is also a promising model that shares several advantages with marine invertebrate eggs, yet has the advantage of being a vertebrate. For example, the zebrafish eggs are reasonably clear optically and can be obtained in quantities suitable for biochemical analysis. They can be fertilized synchronously and will develop rapidly. The zebrafish system also benefits from the fact that the DNA sequence homology with mammals is very high, so that tools developed in mammalian systems can be applied to zebrafish eggs with a reasonable expectation of success. Finally, the potential for genetic analysis of the fertilization process could ultimately provide novel insights into the signaling mechanisms used at fertilization. Despite the above-listed advantages, the zebrafish egg does have some characteristics that complicate biochemical analysis. The relatively large amount of yolk and internal membranous organelles in zebrafish eggs has made detection of relatively low-abundance signal transduction enzymes difficult. The immunoprecipitation steps usually required for detection of Src family kinases and other signaling enzymes do not work well on egg homogenates or detergent extracts of whole zebrafish eggs. Similar problems in the Xenopus oocyte were overcome by subcellular fractionation. The objective of this chapter is to describe recently developed subcellular fractionation techniques that have enabled us to overcome the interference by egg yolk components and successfully quantitate the Fyn tyrosine kinase as well as other PTKs and PTPases. These methods represent a refinement of our previous studies and could be easily adapted to study other low-abundance enzymes present in the membrane fraction of eggs.
Genes / Markers
Figures
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping