![]() ![]() Since overexpression can be associated with aberrant protein localization and interactions ( 9), it is often better to study endogenously expressed proteins. It was used in experiments in SW1736 human anaplastic thyroid and HeLa human cervical cancer cell lines that were made to overexpress NIS ( 8). To our knowledge, except for our studies, PLA has only been used in the field of thyroid research to show dimerization of the sodium-iodide symporter (NIS). For these reasons, PLA is better suited for studies of endogenously expressed proteins in primary cells. Compared to Co-IP, PLA can be scaled down in terms of number of cells and amount of reagents. Since this method uses fluorescent microscopy, information on subcellular location can also be collected. In contrast, PLA is done after fixation, which could capture transient events and preserve low-affinity interactions. Transient, low-affinity interactions, which are common in GPCR signaling, are difficult to detect with Co-IP. For this method to work, interactions between proteins in the complex must be strong enough so that their affinity is maintained while in solution. These binding partners are later identified with Western blotting. An antibody pulls down one protein member and its binding partners. Co-IP is a technique used to identify protein-protein interactions by using target protein-specific antibodies to capture proteins that are bound to a specific target protein. Both PLA and Co-IP depend on antibodies that recognize proteins in the complex. In situ PLA has some advantages over methods such as co-immunoprecipitation (Co-IP). (D) PLA signals look like bright dots on the cell surface. (C) During amplification, fluorescently tagged oligonucleotides bind DNA. (B) PLUS and MINUS DNA probes will ligate and create a DNA circle that becomes a template for rolling circle amplification. In a signalosome, the receptors were shown to be within 40 nm of each other. These antibodies bind to the receptors’ extracellular regions on fixed cells. (A) PLA oligonucleotides PLUS and MINUS are directly conjugated to primary antibodies for TSHR and IGF1R, respectively. ![]() Schematic of Proximity Ligation Assay (PLA) in the TSHR/IGF1R Signalosome. ![]()
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