Epsilon Archive for Student Projects

Abstract

Proximity ligation assay (PLA) is a novel affinity based method used to detect proteins, protein-protein interactions and post-translational modifications. The detection of target molecules is achieved with the help of oligonucleotide labeled antibodies. The oligonucleotides attached on antibodies can be joined by ligation when the conjugates are brought in proximity by binding on the same target molecule (Gullberg et al., 2003). The ligated products can be used as templates in qPCR for quantification. PLA is performed in two approaches, 1) solid surface; where a solid support is used to which a capture antibody is immobilized and target is incubated to capture antibody (Fredriksson et al., 2002). 2) The other approach is solution phase; where there is no solid support, hence no capture antibody is used nor any washing step, and target molecule is incubated directly with detection antibodies. In my project, I have utilized solution phase and solid phase PLA for detection of IGF-1 and detection of phosphorylation on platelet derived growth factor receptor (PDGFβr).

This thesis project aimed to develop an assay for IGF detection and post-translational modification detection in vitro using proximity ligation. My experiment could not establish PLA as a standard assay for detecting IGF-1 and phosphorylated-PDGFβr on solid surface and in solution phase. However, the recombinant PDGFr molecule was successfully detected with solid phase PLA. In this thesis, advantages of PLA and possible reasons for unsuccessful detection of phosphorylated site on PDGFβr are discussed.