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Magalam , Raghavendra, 2014. Detection of IGF-1 and phosphorylated site in PDGFr using proximity ligation assay. Second cycle, A1E. Uppsala: SLU, Dept. of Microbiology

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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.

Main title:Detection of IGF-1 and phosphorylated site in PDGFr using proximity ligation assay
Authors:Magalam , Raghavendra
Supervisor:Blomqvist, Johanna and Kamali Moghaddam, Masood and Yan, Junhong
Examiner:Passoth, Volkmar
Series:Examensarbete / Sveriges lantbruksuniversitet, Institutionen för mikrobiologi
Volume/Sequential designation:2014:9
Year of Publication:2014
Level and depth descriptor:Second cycle, A1E
Student's programme affiliation:None
Department:(NL, NJ) > Dept. of Microbiology
Keywords:PLA, IGF-1, PDGFβr, IgG, recombinant PDGFr, VEGF, solid phase, solution phase
URN:NBN:urn:nbn:se:slu:epsilon-s-3976
Permanent URL:
http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-s-3976
Subjects:Life sciences
Language:English
Deposited On:06 Nov 2014 10:58
Metadata Last Modified:06 Nov 2014 10:58

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