title: Ultrasensitive detection of cellular protein interactions using bioluminescence resonance energy transfer quantum dot-based nanoprobes.
abstract: Sensitive detection of protein interactions is a critical step toward understanding complex cellular processes. As an alternative to fluorescence-based detection, Renilla reniformis luciferase conjugated to quantum dots results in self-illuminating bioluminescence resonance energy transfer quantum dot (BRET-Qdot) nanoprobes that emit red to near-infrared bioluminescence light. Here, we report the development of an ultrasensitive technology based on BRET-Qdot conjugates modified with streptavidin ([BRET-Qdot]-SA) to detect cell-surface protein interactions. Transfected COS7 cells expressing human cell-surface proteins were interrogated with a human Fc tagged protein of interest. Specific protein interactions were detected using a biotinylated anti-human Fc region specific antibody followed by incubation with [BRET-Qdot]-SA. The luciferase substrate coelenterazine activated bioluminescence light emission was detected with an ultra-fast and -sensitive imager. Protein interactions barely detectable by the fluorescence-based approach were readily quantified using this technology. The results demonstrate the successful application and the flexibility of the BRET-Qdot-based imaging technology to the ultrasensitive investigation of cell-surface proteins and protein-protein interactions.
authors.affiliation: Protein Chemistry Department, Genentech, Inc., San Francisco, California 94080, USA.
authors.forename: Gabriel A
authors.initials: GA
authors.lastname: Quiñones
authors.forename: Steven C
authors.initials: SC
authors.lastname: Miller
authors.forename: Sukanta
authors.initials: S
authors.lastname: Bhattacharyya
authors.forename: Daniel
authors.initials: D
authors.lastname: Sobek
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Stephan
mesh_terms: D016475:3T3 Cells; D000818:Animals; D060807:Bioluminescence Resonance Energy Transfer Techniques / Q000379:methods*; D019556:COS Cells; D002522:Chlorocebus aethiops; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D049410:Luciferases, Renilla; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D053758:Nanoparticles; D025941:Protein Interaction Mapping / Q000379:methods*; D011719:Pyrazines / Q000494:pharmacology; D045663:Quantum Dots; D011961:Receptors, Fc / Q000276:immunology; D019809:Streptavidin / Q000737:chemistry
chemical_list: D007093:Imidazoles; D008565:Membrane Proteins; D011719:Pyrazines; D011961:Receptors, Fc; C017144:coelenterazine; D019809:Streptavidin; D049410:Luciferases, Renilla