Extraction and immunoblotting of proteins from embryos.
For many decades, Drosophila has been used as a model system primarily for studies in the fields of genetics and developmental biology. Relatively little attention has been given to the potential of Drosophila as a model system for biochemistry. However, Drosophila embryos as a source for biochemical material offer some unique advantages as compared with cultured cells or tissue samples. For instance, mutant Drosophila embryos can be sorted before protein extraction and compared with wild-type embryos by using green fluorescent protein-marked balancer chromosomes. Studies of this kind can give important information on the effect of a mutation on the biochemical properties of a protein, which cannot be obtained in experiments using cultured cells or conventional tissue samples (1,2). Transgenic Drosophila embryos expressing a tagged version of a protein can be used to isolate and identify interaction partners of the tagged protein from a whole organism rather than from a specific cell line that expresses only a limited set of genes (3,4). Thus, it is the combination of genetics and transgenic approaches that offers unique opportunities for biochemical studies in the fruit fly. In this chapter, I describe methods to extract proteins under denaturing and nondenaturing conditions from embryos, and to perform sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and coimmunoprecipitation.
Wodarz
Andreas
A
Department of Stem Cell Biology, DFG Research Center for Molecular Physiology of the Brain (CMPB), University of Göttingen, Göttingen, Germany.
D000818:Animals; D030801:Animals, Genetically Modified; D015509:Developmental Biology / Q000379:methods*; D029721:Drosophila Proteins / Q000378:metabolism; D004331:Drosophila melanogaster / Q000196:embryology*; D004625:Embryo, Nonmammalian / Q000378:metabolism* / Q000502:physiology*; D018507:Gene Expression Regulation, Developmental; D017344:Genes, Insect; D005821:Genetic Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D015151:Immunoblotting / Q000379:methods*; D008164:Luminescent Proteins / Q000378:metabolism; D008954:Models, Biological; D019076:Transgenes
D029721:Drosophila Proteins; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins