This work details the design and generation of a monomer specific p53 rabbit polyclonal
antibody. The technique involved careful analysis of published Nuclear Magnetic
Resonance(NMR)and X-Ray structures to select an epitope buried in the wildtype tetramer of p53,while exposed in the monomer. The antibody was validated with indirect Enzyme Linked Immunosorbant Assay(ELISA),competition ELISA and western blot. Following these experiments, the antibody, denoted as α-tet,was employed in immunofluorescence (IF)
experiments of cancer cell lines DU145
(prostate carcinoma, p53 P223L and V274F) and A549 (lung carcinoma,p53 wildtype)to determine the localization of monomeric p53. Monomeric p53 was confined to the nucleolus of DU-145 cells. Additional staining of the Golgi apparatus in both cell lines was also observed. However, upon investigation of a p53 null cell line, SKOV-3,the Golgi staining was determined to be a result of cross reactivity with another protein.
Nevertheless, the presence of nucleolar monomeric p53 in DU-145 cells indicates that
monomerization of p53 does occur in this cell line.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OTU.1807/25545 |
| Date | 31 December 2010 |
| Creators | Dann, Cale |
| Contributors | Chakrabartty, Avijit |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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