The Development of a Novel Fluorescence Polarization Drug-Screening Assay for the Interaction Between GIT1 and GRB2

Persistent Link:
http://hdl.handle.net/10150/614109
Title:
The Development of a Novel Fluorescence Polarization Drug-Screening Assay for the Interaction Between GIT1 and GRB2
Author:
Gonzales, Jared; Vaillancourt, Richard
Affiliation:
College of Pharmacy, The University of Arizona
Issue Date:
2015
Rights:
Copyright © is held by the author.
Collection Information:
This item is part of the Pharmacy Student Research Projects collection, made available by the College of Pharmacy and the University Libraries at the University of Arizona. For more information about items in this collection, please contact Jennifer Martin, Associate Librarian and Clinical Instructor, Pharmacy Practice and Science, jenmartin@email.arizona.edu.
Publisher:
The University of Arizona.
Abstract:
Objectives: To develop an assay to permit the identification of compounds that can inhibit the interaction between GIT1 and the amino-terminal SH3 domain (SH3-N) of GRB2. Methods: The GIT1 protein was expressed in Sf9 insect cells and purified using Talon resin beads. The SH3-N domain of GRB2 was expressed in the E. coli strain, BL21(DE3)pLysS, and purified using glutathione resin beads. The SH3-N domain was fluorescently tagged on cysteine 32 using Cyanine 3 maleimide. The fluorescence of the assay was measured by using a plate reader with excitation wavelength of 555 nm and emission wavelength of 570 nm. Results: The GIT1 protein was expressed in Sf9 cells and purified using the Talon beads. The SH3-N domain of GRB2 was expressed in BL21 cells and purified from the glutathione resin beads. The SH3-N domain was cleaved from GST by using thrombin, which was engineered into the GST fusion protein and were fluorescently labeled using Cyanine 3 maleimide. Conclusions: The fluorescence polarization assay that will detect the interaction between GIT1 and the SH3-N domain of GRB2 is still under development, but it has progressed towards completion since both components of the assay are in hand.
Description:
Class of 2015 Abstract
Keywords:
drug-screening; GIT1; GRB2; fluorescence
Advisor:
Vaillancourt, Richard

Full metadata record

DC FieldValue Language
dc.contributor.advisorVaillancourt, Richarden
dc.contributor.authorGonzales, Jareden
dc.contributor.authorVaillancourt, Richarden
dc.date.accessioned2016-06-22T16:34:49Z-
dc.date.available2016-06-22T16:34:49Z-
dc.date.issued2015-
dc.identifier.urihttp://hdl.handle.net/10150/614109-
dc.descriptionClass of 2015 Abstracten
dc.description.abstractObjectives: To develop an assay to permit the identification of compounds that can inhibit the interaction between GIT1 and the amino-terminal SH3 domain (SH3-N) of GRB2. Methods: The GIT1 protein was expressed in Sf9 insect cells and purified using Talon resin beads. The SH3-N domain of GRB2 was expressed in the E. coli strain, BL21(DE3)pLysS, and purified using glutathione resin beads. The SH3-N domain was fluorescently tagged on cysteine 32 using Cyanine 3 maleimide. The fluorescence of the assay was measured by using a plate reader with excitation wavelength of 555 nm and emission wavelength of 570 nm. Results: The GIT1 protein was expressed in Sf9 cells and purified using the Talon beads. The SH3-N domain of GRB2 was expressed in BL21 cells and purified from the glutathione resin beads. The SH3-N domain was cleaved from GST by using thrombin, which was engineered into the GST fusion protein and were fluorescently labeled using Cyanine 3 maleimide. Conclusions: The fluorescence polarization assay that will detect the interaction between GIT1 and the SH3-N domain of GRB2 is still under development, but it has progressed towards completion since both components of the assay are in hand.en
dc.language.isoen_USen
dc.publisherThe University of Arizona.en
dc.rightsCopyright © is held by the author.en
dc.subjectdrug-screeningen
dc.subjectGIT1en
dc.subjectGRB2en
dc.subjectfluorescenceen
dc.titleThe Development of a Novel Fluorescence Polarization Drug-Screening Assay for the Interaction Between GIT1 and GRB2en_US
dc.typetexten
dc.typeElectronic Reporten
dc.contributor.departmentCollege of Pharmacy, The University of Arizonaen
dc.description.collectioninformationThis item is part of the Pharmacy Student Research Projects collection, made available by the College of Pharmacy and the University Libraries at the University of Arizona. For more information about items in this collection, please contact Jennifer Martin, Associate Librarian and Clinical Instructor, Pharmacy Practice and Science, jenmartin@email.arizona.edu.en
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