Acquisition of host-derived proteins possessing key regulatory function is a hallmark of Borrelia burgdorferi, and an important step to successfully infect the human host, inhibiting activation of complement as innate immunity’s first line of defense. Hence, the identification and characterization of interacting ligands is a prerequisite to gain deeper insights into the molecular principles of how spirochetes overcome the detrimental effects of complement. Far western blotting enables the detection of protein-protein interactions in vitro using cell lysates containing the prey proteins and purified complement proteins or human serum as a source for soluble complement proteins as bait proteins. Here, the methodology for the detection and characterization of Borrelia-derived proteins interacting with complement regulator Factor H is described, including the preparation of whole cell lysates, the separation of proteins by Tris-Tricine SDS-PAGE, the transfer of the proteins to nitrocellulose membranes, and the detection of Factor H-interacting proteins by Far western.
BorreliaSpirochete Lyme disease Complement Factor H Far western Tricine SDS-PAGE
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