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Notch-Ligand Binding Assays in Drosophila Cells

  • Aiguo Xu
  • Kenneth D. IrvineEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1187)

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 cells 

Notes

Acknowledgments

Research in KDIs lab is supported by the Howard Hughes Medical Institute.

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Primera Analytical Solutions CorpPrincetonUSA
  2. 2.Howard Hughes Medical InstituteRutgers UniversityPiscatawayUSA
  3. 3.Waksman Institute and Department of Molecular Biology and BiochemistryRutgers UniversityPiscatawayUSA

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