#31: Antibody-based Detection of GNAQ Protein in a Zebrafish Model of Uveal Melanoma — Presentation Detail — Celebration of Scholars

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#31: Antibody-based Detection of GNAQ Protein in a Zebrafish Model of Uveal Melanoma

Name: Neva Bergemann
Major: Biology
Hometown: Sussex, WI
Faculty Sponsor:
Other Sponsors:
Type of research: Independent research

Abstract

Uveal melanoma is an aggressive cancer that affects melanocytes within the uvea of the eye. Uveal melanoma can be extremely deadly due to its likelihood of metastasis. Successful therapies common to other forms of melanoma have not proven effective for uveal melanoma. Ultimately, this highlights the necessity for a deeper understanding of uveal melanoma development and progression so that functional therapies can be developed and applied. One of the driving oncogenes within uveal melanoma is GNAQ, which is mutated and codes for a hyperactive G-alpha protein in approximately 93% of patients. Studying GNAQ-driven uveal melanoma through the lens of a model organism is essential for the eventual development of therapeutic targets. Zebrafish are excellent cancer models due to their genetic similarity to humans, ease of genetic manipulation, and their regenerative properties. Transgenic fish that express the human mutated GNAQ gene display melanocyte-specific abnormalities and discoloration, which makes distinguishing between the GNAQ transgenic and wild type zebrafish relatively easy. However, there are gaps in this model as there is currently no effective way to measure the expression of GNAQ protein. Our research has focused on determining the ideal number of zebrafish caudal fins and embryos that will allow for measurement of the GNAQ protein within GNAQ and wild type zebrafish. Once the optimized ratio of 1 caudal fin to 100 µL of protease inhibitor was identified, a BCA assay was performed to quantify the amount of protein present in the tissue extracts. Subsequent methods included an SDS-PAGE and a Western blot to detect and compare the levels of the GNAQ protein in transgenic and wild type zebrafish. This analysis identified the mouse anti-human GNAQ/11/14 antibody (Santa Cruz, catalog # sc-365906) as a functional antibody to detect GNAQ in wild type and transgenic zebrafish. Future directions can include testing additional protein dilutions, antibody concentrations, and testing other antibodies for the optimal measurement of the GNAQ protein.

Poster file