From GFP to pFLAG: Confirmation of Intracellular Location of KIAA1946
Location
CSU 253/4/5
Start Date
5-4-2010 10:00 AM
End Date
5-4-2010 12:00 PM
Student's Major
Biological Sciences
Student's College
Science, Engineering and Technology
Mentor's Name
Geoffrey Goellner
Mentor's Department
Biological Sciences
Mentor's College
Science, Engineering and Technology
Description
Although significant advances have been made in deciphering the human proteome, there are still close to 8,000 genes whose functions are still completely unknown. In this study, we have attempted to determine the cellular location of one such novel gene product- KIAA1946. Indeed, very little is known regarding KIAA1946’s normal function in cells. However, we do know that it is likely expressed in the nervous system, and it has a polyglutamine region in its primary amino acid sequence- two interesting features as other polyglutamine proteins have been linked to severe neurodegenerative diseases such as Huntington’s chorea. Previous studies (using green fluorescent protein- GFP) in our laboratory have shown that KIAA1946 likely localizes to cytoplasmic vesicles. In the current study, we have attempted to confirm this initial data by using standard molecular biology tools (such as restriction digestion and ligation) to sub-clone KIAA1946 into a vector containing the FLAG epitope (commercial antibodies are available against FLAG). We checked for proper cloning of our FLAG-KIAA1946 fusion protein using restriction digestion coupled to agarose gel electrophoresis- and also officially confirmed our clone integrity using DNA sequencing. Next, we transfected our new FLAG-KIAA1946 clone into tissue culture animal cells, and allowed several days for the protein to appropriately localize within the cells. Finally, we employed immunofluorescence microscopy using antibodies against the FLAG moiety to ―visualize‖ the location of our fusion protein. Data regarding KIAA1946’s location within cells will provide us invaluable insight regarding the normal cellular function of this novel polyglutamine protein.
From GFP to pFLAG: Confirmation of Intracellular Location of KIAA1946
CSU 253/4/5
Although significant advances have been made in deciphering the human proteome, there are still close to 8,000 genes whose functions are still completely unknown. In this study, we have attempted to determine the cellular location of one such novel gene product- KIAA1946. Indeed, very little is known regarding KIAA1946’s normal function in cells. However, we do know that it is likely expressed in the nervous system, and it has a polyglutamine region in its primary amino acid sequence- two interesting features as other polyglutamine proteins have been linked to severe neurodegenerative diseases such as Huntington’s chorea. Previous studies (using green fluorescent protein- GFP) in our laboratory have shown that KIAA1946 likely localizes to cytoplasmic vesicles. In the current study, we have attempted to confirm this initial data by using standard molecular biology tools (such as restriction digestion and ligation) to sub-clone KIAA1946 into a vector containing the FLAG epitope (commercial antibodies are available against FLAG). We checked for proper cloning of our FLAG-KIAA1946 fusion protein using restriction digestion coupled to agarose gel electrophoresis- and also officially confirmed our clone integrity using DNA sequencing. Next, we transfected our new FLAG-KIAA1946 clone into tissue culture animal cells, and allowed several days for the protein to appropriately localize within the cells. Finally, we employed immunofluorescence microscopy using antibodies against the FLAG moiety to ―visualize‖ the location of our fusion protein. Data regarding KIAA1946’s location within cells will provide us invaluable insight regarding the normal cellular function of this novel polyglutamine protein.
Recommended Citation
Becker, Anita and Jessica Appel. "From GFP to pFLAG: Confirmation of Intracellular Location of KIAA1946." Undergraduate Research Symposium, Mankato, MN, April 5, 2010.
https://cornerstone.lib.mnsu.edu/urs/2010/poster-session-A/12
