Detection of intracellular ADAMTS13, a secreted zinc-metalloprotease, via flow cytometry.

ABSTRACT: ADAMTS13 is a secreted metalloprotease that cleaves von Willebrand Factor multimers in order to maintain proper homeostasis. A severe deficiency in ADAMTS13 triggers a disorder known as thrombotic thrombocytopenic purpura. At present, ADAMTS13 expression levels are determined by immunoblotting. We established a flow cytometry methodology to detect intracellular ADAMTS13 in liver and kidney cells using a polyclonal antibody, BL154G, and several monoclonal antibodies previously used to detect ADAMTS13 by immunoblotting. The results were validated using confocal microscopy, immunoblotting, and an activity assay (FRETS-VWF73). We show that labeling ADAMTS13 with specific antibodies and detection by flow cytometry yields results that are comparable with previously established methods for ADAMTS

SUBMITTER: Geetha S 

PROVIDER: S-EPMC2778596 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature