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Capitolo VI. Materiali e Metod

6.8 Coimmunoprecipitazione

L’immunoprecipitatazione delle proteine: BAG3, SNAP-25, Sintaxina, Paxillina e FAK dai lisati delle B TC 6, è stata effettutata utilizzanzo la resina della SANTA Cruz Biotechnology, inc ( IP matrix mouse). Brevemente: 45ul di resina sono stati incubati per 1h a 4° C, in agitazione continua, con 3ug dell’anticorpo anti BAG3 (AC-1), anti-SNAP-25, anti-Sintaxina, anti-paxillina e anti- Fak. La resina è stata inseguito lavata con PBS + 1% Tween per due volte e centrifugata 1’ a 13.000 rpm 4°C. Sulla resina attivata sono state caricate 300ug di proteine ottenute dai lisati delle βTC- 6; resina e lisati sono stati incubati per 2h a 4° C, in agitazione continua. Si è in seguito proceduto a lavare la resina tre volte con RIPA BUFFER (Tris / HCl ph 7.6 50mM, NaCl150mM, EDTA 4mM, NaF 10mM, Sodio pirofosfato 10mM, 1% NP-40, 1% Sodio deossicolato) centrifugando ogni volta 1’ a 13000 rpm, 4°C. Infine la resina è stata eluita con 45 ul di Laemmly Buffer 2X e conservata a - 20°C. I campioni sono stati successivamente processati per western blotting.

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Ringraziamenti

Vorrei ringraziare tutti coloro che hanno reso possibile la realizzazione di questa tesi. Primo fra tutti ringrazio il Prof. Gianfranco Tajana per avermi guidato verso la ricerca

e trasmesso l’amore per essa.

La Prof.ssa Maria Caterina Turco per avermi dato la possibilità di svolgere il mio progetto di ricerca ed avermi seguita durante questi tre anni di dottorato. La dott.ssa Michelina Festa per i suoi consigli e la sua umanità, per avermi insegnato

a mettere in atto le mie idee e per essermi sempre stata vicina, spronandomi nei momenti di difficoltà.

La Dott.ssa Alessandra Rosati per la sua disponibilità e per avermi seguito durante la fase di stesura della tesi.

Maria Caterina De Rosa, Margot De Marco, Raffaella D’Auria, Luana Guerriero, Anna Gorrasi, Roberta Esposito, Gaetano Pierro, Roberta Cotugno,Mariella Caputo, Anna Basile, Antonia Falco, Morena D’Avenia, Francesca Isabella De Simone per aver reso piacevole e familiare l’ambiente in cui ho lavorato in questo tre anni.

Ringrazio infine la mia famiglia e Valerio che ha sempre creduto in me e mi è stato accanto nei momenti di difficoltà.

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