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Title:The Development of an Assay for Identifying the Presence of Inorganic Polyphosphate in a Sample
Author(s):McClintock, Andrew S.
Contributor(s):Morrissey, James H.
Subject(s):polyphosphate
PPXbd
PVDF
biotin
chemiluminescence
Abstract:In hematology, recent studies have identified inorganic polyphosphate (polyP) as a molecule that has significant consequences in the blood coagulation cascade. As polyP becomes increasingly important in hematology, therefore, it is necessary to develop simplified methods of polyP detection to make it possible to determine which tissues use this molecule regularly, as this information could lead to improved knowledge regarding polyP and its mechanisms of action. Furthermore, it would be particularly helpful if a protocol were developed that could directly detect non-labeled polyP, as the introduction of covalent modifications to mediate detection can often be time-consuming and difficult, especially for polyP samples that are not covalently modified in vivo. In these experiments, protocols involving the identification of non-labeled polyP through the attachment of these molecules onto a membrane such as PVDF were conducted using a detection scheme involving the identification of biotin with streptavidin-HRP, followed by the use of a substrate with enhanced chemiluminescent properties (ECL). Results showed that the non-specific binding of biotinylated PPXbd to many widely used membranes poses a significant obstacle in this protocol, so much of this research entailed the search of a blocking set-up that reduced this non-specific binding. While no definitive answers were found, this research still has the potential to become something that can work in the long-term if a biotinylated molecule or protein that binds to polyP with high affinity without non-specifically binding to a membrane can be identified, eventually leading to simplified methods of identifying polyP in vitro.
Issue Date:2017
Genre:Dissertation / Thesis
Type:Text
URI:http://hdl.handle.net/2142/96058
Rights Information:Copyright 2017 Andrew S. McClintock
Date Available in IDEALS:2017-05-16


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