Notch Signaling pp 277-284 | Cite as
Notch-Ligand Binding Assays in Drosophila Cells
Abstract
Activation of the Drosophila transmembrane receptor protein Notch is induced by association with its transmembrane ligands, Delta and Serrate. The ability to assay binding between Notch and its ligands has been essential for characterizing the influence of posttranslational modifications, such as glycosylation, as well as for characterizing structural motifs involved in receptor–ligand interactions. We describe here a simple, widely used method for assaying receptor–ligand binding. This method involves expression of soluble forms of either Notch or its ligands, comprising the extracellular domains fused to an easily assayed tag, the enzyme alkaline phosphatase. These soluble proteins are then incubated with their binding partners, either as transmembrane proteins expressed on the surface of cultured cells or as extracellular protein domains attached to agarose beads. After washing, the amount of bound protein can be readily assayed by measuring alkaline phosphatase activity.
Key words
Notch Serrate Delta Receptor Ligand Binding Alkaline phosphatase S2 cellsNotes
Acknowledgments
Research in KDIs lab is supported by the Howard Hughes Medical Institute.
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