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Protein Interaction Essay
BIOC2291 - Fundamentals of Molecular Biology
5 Pages • Essays / Projects • Year: Pre-2021
Proximity ligation assay (PLA) is a technique designed for investigating protein activity in cell such as post translation modification and protein-protein interaction that regulate the cell-signalling pathway. This assay targets epitopes on the two interacting proteins by using two primary antibodies raised in different animal species. A pair of PLA probes (each consisting of a species-specific secondary antibody and a conjugated oligonucleotide) will subsequently bind to their respective primary antibodies. This combination provides a highly sensitive and specific method to detect protein-based events. When the targeted epitopes are within a distance of 30-40nm, the oligonucleotides of the PLA probes are brought in proximity. They can then be joined by connector oligonucleotides via enzymatic ligation to form a circular DNA molecular. Next, the circular DNA molecular is amplified through rolling circle amplification (RCA) by using oligonucleotide in one of the PLA probes as a primer, which makes single stranded DNA fold into a bundle and hundredfold replication of DNA circle produced. Fluorescent probes conjugated oligonucleotides hybridize with RCA products to produce signals that can easily be visualized under fluorescence microscope. Image analysis is used to measure the quality of target protein (Sigrun M. Gustafsdottira, 2005). This assay has successfully been proved the interaction of SMAD1/2/3 and the SMAD4 protein
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