Expression of GLYATL1 Plasmid in Transfected MCF7 and HEK293FT Cell Lines
School Name
South Carolina Governor's School for Science and Mathematics
Grade Level
12th Grade
Presentation Topic
Cell and Molecular Biology
Presentation Type
Mentored
Abstract
Luminal A breast cancers, characterized by high estrogen-receptor expression, can be treated with endocrine therapies that inhibit estrogen binding to receptors. However, up to 40% of patients are resistant or develop resistance to these therapies. In the lab, luminal A breast cancer cell lines are studied to characterize molecular changes upon resistance. Sequencing revealed overexpression of GLYATL1 gene in resistant cells. There is no available antibody that targets the GLYATL1 proteins, so an antibody must be developed to better test the function of GLYATL1 in resistant cells. To test the specificity of the developed antibody, plasmid containing a sequence of GLYATL1 and a flag tag was transfected into the MCF7 and HEK293FT cells. The protein expression was tested on a Western Blot. Since the flag tag is small (1 kDa), a band is expected at 35 kDa. However, this transfection was found to be unsuccessful. In a second run, plasmids containing GLYATL1-GFP and GLYATL1-flag were transfected in HEK293FT cells. A band was detected at 70 kDa, which was the expected height given the GFP tag’s size of 27 kDa. Additionally, a band was detected at 35 kDA indicating the flag tagged proteins. The knowledge of the GLYATL1 characteristic protein bands on Western Blot will be used to test the specificity of the developed antibody. This antibody allows for specific assays to be performed to better understand the function of GLYATL1 in therapy resistant luminal A breast cancer cells.
Recommended Citation
O’Brien, Mary, "Expression of GLYATL1 Plasmid in Transfected MCF7 and HEK293FT Cell Lines" (2024). South Carolina Junior Academy of Science. 441.
https://scholarexchange.furman.edu/scjas/2024/all/441
Location
RITA 271
Start Date
3-23-2024 10:45 AM
Presentation Format
Oral Only
Group Project
No
Expression of GLYATL1 Plasmid in Transfected MCF7 and HEK293FT Cell Lines
RITA 271
Luminal A breast cancers, characterized by high estrogen-receptor expression, can be treated with endocrine therapies that inhibit estrogen binding to receptors. However, up to 40% of patients are resistant or develop resistance to these therapies. In the lab, luminal A breast cancer cell lines are studied to characterize molecular changes upon resistance. Sequencing revealed overexpression of GLYATL1 gene in resistant cells. There is no available antibody that targets the GLYATL1 proteins, so an antibody must be developed to better test the function of GLYATL1 in resistant cells. To test the specificity of the developed antibody, plasmid containing a sequence of GLYATL1 and a flag tag was transfected into the MCF7 and HEK293FT cells. The protein expression was tested on a Western Blot. Since the flag tag is small (1 kDa), a band is expected at 35 kDa. However, this transfection was found to be unsuccessful. In a second run, plasmids containing GLYATL1-GFP and GLYATL1-flag were transfected in HEK293FT cells. A band was detected at 70 kDa, which was the expected height given the GFP tag’s size of 27 kDa. Additionally, a band was detected at 35 kDA indicating the flag tagged proteins. The knowledge of the GLYATL1 characteristic protein bands on Western Blot will be used to test the specificity of the developed antibody. This antibody allows for specific assays to be performed to better understand the function of GLYATL1 in therapy resistant luminal A breast cancer cells.
