Identification of reagents intended to the study of the long isoform of the NF-YA transcription factor

• Autor(a) principal: Miranda, Bárbara Filipa Teixeira de Araújo
• Data de Publicação: 2021
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/31016
• topic: Transcription factor; Nuclear factor Y; NF-YA; Alternative splicing; NF-YA long

Transcription factors are proteins involved and vital to several cellular processes and cell identity. The binding of these proteins to the specific DNA elements is what makes it so important, regulating the transcriptome of all cells. The nuclear factor Y is a protein classified as a pioneer TF, binding to the CCAAT box promoter element. This NF is composed by three different and preserved subunits NF-YA, NF-YB and NF-YC. NF-YA is the regulatory subunit and is composed by 347 aminoacids in its long isoform (NF-YAl). The NF-YA gene expression generates in fact, by alternative splicing, two main isoforms, differing in 28 aminoacids, corresponding to the exon 3. Due to the essential role this nuclear factor plays in many cellular processes it is crucial the deepening of the knowledge about its physiological and pathological functions, in particular regarding the differential expression of the different isoforms of NF-YA. In this project, which initially started after two rabbits were immunized with NF-YAl, we performed experiments to obtain NF-YA long specific antibody reagents, essential to distinguish both isoforms in further experiments. This project aims included the production and purification of recombinant proteins for the subsequent affinity purification of the specific antibodies. To accomplish the main objective, the project started with the preparation of bacterial competent cells for transformation with the relevant expression plasmids, followed by setting the induction and purification protocols for protein production. After the required amount of protein was obtained, we proceeded to the final steps of the project with experiments of western blot and affinity chromatography of rabbit sera. The elution fractions obtained by affinity chromatography purifications were subsequently tested and analysed by western blot, to verify the specificity of NF-YAl antibodies. The principal aim was achieved with success, having as a final positive result in two elution fractions, corresponding to a specific pH of the affinity chromatography, which included antibodies specific to the long isoform of NF-YA.