Pubblicazioni Aperte DIgitali Sapienza > Scienze biochimiche "A. Rossi Fanelli" > BIOCHIMICA >

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Title: Involvement of ERp57 in signal transduction
Tutor: Turano, Carlo
Keywords: ERp57
Issue Date: 1-Dec-2009
Research interests: My research interests are mainly focused on biochemistry and cell biology, particularly for what concerns signal transduction mechanisms and intracellular trafficking. I am also interested in molecular biology, especially in protein-protein and protein-DNA interactions, as well as in the control of gene expression.
Skills short description: I have attended to the growth of several cancer or immortalized cell lines, both in adhesion or in suspension. The maintenance of cell cultures has required also control procedures to exclude any possible contamination, by means of PCR or staining with fluorescent dyes. I have also performed MTT or MTS assays to assess cell viability. I often have performed the RNA interference procedure in order to silence the expression of a protein. This recent technique consists in the administration of a specific short interfering RNA (siRNA) directly to the cells by means of a transfection reagent. In order to have the highest silencing efficiency, it is fundamental to regulate the quantity of seeded cells with the amount of the administered siRNA. The choice of the proper transfection reagent is indeed very important, in order to minimize possible toxic side effects. To evaluate the rate of silencing, I have analyzed the silenced cells both at mRNA and protein levels, by RT-PCR or SDS-PAGE and Western blotting respectively. I have had the opportunity to perform many immunofluorescence experiments, on cells grown directly on coverslips or transferred to slides by cytospin. I have performed also double immunofluorescence to visualize two antigens in the same cell, which has implied the accurate selection of antibodies and fluorophores. I have analyzed the cells by means of wide-field or confocal fluorescence microscopy. I have performed both co-immunoprecipitations (CoIP) and chromatin immunoprecipitations (ChIP). In the first case the aim is to investigate the possible direct interaction of two proteins; in the second one the interaction of a protein with DNA. I have used different cross-linking reagents, according to the characteristics of the interaction. Furthermore, I have performed fluorescence-based assays to determine the affinity constant of ligand-protein complexes.
Personal skills keywords: Cell cultures
RNA interference
immunofluorescence microscopy

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