Generation of Rabbit and Chicken Polyclonal Antibodies
Location
CSU Ballroom
Start Date
21-4-2008 1:00 PM
End Date
21-4-2008 3:00 PM
Student's Major
Biological Sciences
Student's College
Science, Engineering and Technology
Mentor's Name
Marilyn C. Hart
Mentor's Department
Biological Sciences
Mentor's College
Science, Engineering and Technology
Description
Actin, a cytoskeletal component of all eukaryotic cells, plays an important role in diverse cell functions, including maintaining cell shape and contributing to cell motility. Actin filament length and stability is regulated by a variety of accessory proteins including actin capping protein (CP). In vertebrates, three alpha isoforms (α1, α2, α3) and three beta isoforms (β1, β2, β3) have been identified. We hypothesize that the alpha isoforms have distinct functions in tissues and cells which suggests that the proteins have different localization patterns. To evaluate the expression of the alpha proteins, we are generating alpha isoforms' specific antibodies which will be used in future localization studies. Because alpha isoform antibodies do not exist, I am preparing two new polyclonal anti-mouse CP sera, one generated in chicken and one generated in rabbit, which will allow for double localization studies. The immunogens were peptides for mouse CP α1and CP α2, one specific for mouse al and one specific for mouse α2. Fusion protein constructs were prepared in pGEX-6, a glutathione S transferase vector. The protein were expressed In E. coli and purified by affinity chromatography. The protein concentration was determined by Bradford analysis and used as an immunogen in both chicken and rabbit. We have determined the titer of the production antibodies using Western Blot analysis. The α1 antibodies, generated in chicken, have a reactive titer of 10-6. α2 antibody production in rabbit is underway.
Generation of Rabbit and Chicken Polyclonal Antibodies
CSU Ballroom
Actin, a cytoskeletal component of all eukaryotic cells, plays an important role in diverse cell functions, including maintaining cell shape and contributing to cell motility. Actin filament length and stability is regulated by a variety of accessory proteins including actin capping protein (CP). In vertebrates, three alpha isoforms (α1, α2, α3) and three beta isoforms (β1, β2, β3) have been identified. We hypothesize that the alpha isoforms have distinct functions in tissues and cells which suggests that the proteins have different localization patterns. To evaluate the expression of the alpha proteins, we are generating alpha isoforms' specific antibodies which will be used in future localization studies. Because alpha isoform antibodies do not exist, I am preparing two new polyclonal anti-mouse CP sera, one generated in chicken and one generated in rabbit, which will allow for double localization studies. The immunogens were peptides for mouse CP α1and CP α2, one specific for mouse al and one specific for mouse α2. Fusion protein constructs were prepared in pGEX-6, a glutathione S transferase vector. The protein were expressed In E. coli and purified by affinity chromatography. The protein concentration was determined by Bradford analysis and used as an immunogen in both chicken and rabbit. We have determined the titer of the production antibodies using Western Blot analysis. The α1 antibodies, generated in chicken, have a reactive titer of 10-6. α2 antibody production in rabbit is underway.
Recommended Citation
Kastenschmidt, Jenna. "Generation of Rabbit and Chicken Polyclonal Antibodies." Undergraduate Research Symposium, Mankato, MN, April 21, 2008.
https://cornerstone.lib.mnsu.edu/urs/2008/poster-session-B/12
