Univerzita PardubiceFakulta chemicko- technologická

Univerzita Pardubice

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English

Publikace detail

HPLC-separation of Tau protein fragments for subsequent analysis of immunoreactive domains

Autoři: Jankovičová Barbora | Link Marek | Kanďár Roman | Drábková Petra | Bartoš Aleš | Slováková Marcela | Korecká Lucie | Stulík Jiří | Bílková Zuzana

Rok: 2012

Druh publikace: ostatní - přednáška nebo poster

Strana od-do: nestránkováno

Tituly:

Jazyk	Název	Abstrakt	Klíčová slova
eng	HPLC-separation of Tau protein fragments for subsequent analysis of immunoreactive domains	Tau protein is considered to be important for maintaining the stability of axonal microtubules and it is a major component of abnormal intraneuronal aggregates observed in numerous tauopathies, including Alzheimer's disease (AD). Following neuronal damage, Tau protein is released into the extracellular space and might occur in body fluids, such as cerebrospinal fluid (CSF) or blood. Consequently anti-Tau antibodies are present in multifold higher levels in the serum compared to the CSF of AD patients. The levels and the avidities of anti-Tau antibodies were already investigated. We focused in this work on searching for regions of Tau protein molecule immunoreactive with these anti-Tau antibodies. Recombinant human Tau protein-441 was firstly chemically fragmented using cyanogen bromide (CNBr), which hydrolyzes peptide bonds at the C-terminus of methionine residues and thus provides six specific Tau-fragments. Efficiency of fragmentation was determined by SDS-PAGE a MALDI-TOF-MS analysis. Subsequently Tau-fragments were separated by RP-HPLC technique. Setting up a gradient conditions and composition of mobile phases were optimised. Collected fractions were analyzed off-line by MALDI-TOF-MS. Finally the immunoreactivity of the eluted HPLC-fractions was tested using dot-blot combined with anti-Tau antibodies. Comparative analysis with native Tau-protein isolated from CSF of AD patients will come after. We suppose the immunoreactivity can be changed as a consequence of modified post-translational modifications as phosphorylation or glycosylation. Identified Tau-fragments reacting with anti-Tau antibodies have a potential to be applied consequently in clinical practice either for AD diagnosis or for the therapeutic purpose as peptide-based vaccination.	Tau-protein; HPLC-separation; immunoreactivity; CNBr fragmentation