Class of 2015 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.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/614109 |
| Date | January 2015 |
| Creators | Gonzales, Jared, Vaillancourt, Richard |
| Contributors | Vaillancourt, Richard, College of Pharmacy, The University of Arizona |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | en_US |
| Detected Language | English |
| Type | text, Electronic Report |
| Rights | Copyright © is held by the author. |
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