Alkaline phosphatase fusion proteins as affinity probes for protein localization studies.
The receptor affinity probe or receptor alkaline phosphatase (RAP) staining method uses soluble protein ectodomains fused to secreted placental alkaline phosphatase to locate ectodomain binding sites within cells or tissues. We have used this approach to identify expressing cells in tissue culture, in tissue sections, or in whole-mount embryos. The RAP method is especially useful in situations where a reliable monoclonal antibody is not available or if an orphan receptor is the focus of the study. The technique permits localization of both receptors and ligands and is readily quantifiable for cell-surface binding assays. Soluble ectodomain placental alkaline phosphatase fusion proteins are therefore highly sensitive reagents that permit the direct localization of available binding sites through simple chromogenic assays without purification, radioactive labeling, or secondary reagents.
Brennan
Caroline
C
School of Biological Sciences, Queen Mary, University of London, UK. c.h.brennan@qmul.ac.uk
Fabes
Jez
J
D016475:3T3 Cells; D000345:Affinity Labels / Q000378:metabolism*; D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004622:Embryo, Mammalian; D004625:Embryo, Nonmammalian; D036386:Ephrin-A5 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D036388:Ephrin-B2 / Q000096:biosynthesis / Q000235:genetics; D005306:Fertilization / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D007668:Kidney / Q000166:cytology; D051379:Mice; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D036081:Receptors, Eph Family / Q000096:biosynthesis / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D015027:Zebrafish
D000345:Affinity Labels; D018076:DNA, Complementary; D036386:Ephrin-A5; D036388:Ephrin-B2; D011993:Recombinant Fusion Proteins; D036081:Receptors, Eph Family; D000469:Alkaline Phosphatase