Western Blotting For Extracellular Vesicles
Document Type
Event
Faculty Mentor
Larisa Ryzhova
Abstract
The Liaw Lab is researching extracellular vesicles (EVs) and the effect they have on induced pluripotent stem cell (iPSC) cardiomyocytes. EVs are vesicles composed of a phospholipid bilayer, released from cells, that hold proteins, lipids, RNA, and other signaling factors. EVs move this cargo out of the cell, enter the bloodstream or directly signal other cells. EVs are of great interest medically because they have the potential to diagnose or treat certain conditions by carrying disease associated signaling factors. To further study EVs, the Liaw Lab uses rab27a global knockout mice. Rab27a protein is a small GTPase protein necessary for multivesicular endosome docking to the plasma membrane, associated with the release of EVs. Global knock out of the rab27a gene alters the release of EVs from all cells, allowing the lab to compare EV release and activity between knock out and wild type mice. The ultimate goal of this project is to apply the effects of circulating EVs to cardiomyocyte cells. However, EVs are similar in size to other particles present in blood plasma. Therefore this project focuses on the optimization of Isolation and quantification techniques needed to confirm the presence and quality of EVs. These techniques include Size exclusion chromatography (SEC), nanoparticle tracking analysis (NTA), bichonic acid (BCA) assay, sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS PAGE), and western blotting. Western blotting focuses on probing for common tetraspannin proteins CD63 and CD9, associated with EVs. The current focus of this project is to determine if EV isolations are dominated by platelet EVs. This process will further confirm the presence of EVs and their origin. Once the methods of isolation and characterization are fully optimized, the lab plans to use isolated EVs and observe their effect on cardiomyocytes through in vitro iPSC cardiomyocyte contractility assay.
Western Blotting For Extracellular Vesicles
The Liaw Lab is researching extracellular vesicles (EVs) and the effect they have on induced pluripotent stem cell (iPSC) cardiomyocytes. EVs are vesicles composed of a phospholipid bilayer, released from cells, that hold proteins, lipids, RNA, and other signaling factors. EVs move this cargo out of the cell, enter the bloodstream or directly signal other cells. EVs are of great interest medically because they have the potential to diagnose or treat certain conditions by carrying disease associated signaling factors. To further study EVs, the Liaw Lab uses rab27a global knockout mice. Rab27a protein is a small GTPase protein necessary for multivesicular endosome docking to the plasma membrane, associated with the release of EVs. Global knock out of the rab27a gene alters the release of EVs from all cells, allowing the lab to compare EV release and activity between knock out and wild type mice. The ultimate goal of this project is to apply the effects of circulating EVs to cardiomyocyte cells. However, EVs are similar in size to other particles present in blood plasma. Therefore this project focuses on the optimization of Isolation and quantification techniques needed to confirm the presence and quality of EVs. These techniques include Size exclusion chromatography (SEC), nanoparticle tracking analysis (NTA), bichonic acid (BCA) assay, sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS PAGE), and western blotting. Western blotting focuses on probing for common tetraspannin proteins CD63 and CD9, associated with EVs. The current focus of this project is to determine if EV isolations are dominated by platelet EVs. This process will further confirm the presence of EVs and their origin. Once the methods of isolation and characterization are fully optimized, the lab plans to use isolated EVs and observe their effect on cardiomyocytes through in vitro iPSC cardiomyocyte contractility assay.
