Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos.
By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-β receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype. In the first step, embryo protein extracts are prepared and used to generate125I-TGF-β2-TGFBR3 complexes that are purified by immunoprecipitation. Later, these complexes are covalently cross-linked and revealed using SDS-PAGE separation and autoradiography detection. This technique requires the availability of a labeled ligand for, and a specific antibody against, the receptor to be detected, and shall be easily adapted to identify any growth factor or cytokine receptor that meets these requirements.
Cell and Developmental Biology, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México.
Tonatiuh
T
Molina-Villa
Cell and Developmental Biology, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México.
Valentín
V
Mendoza
Cell and Developmental Biology, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México; fcasilla@ifc.unam.mx.
Fernando
F
López-Casillas
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