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@article{1227600,
author = {Němcová, Kateřina and Šebest, Peter and Havran, Luděk and Orság, Petr and Fojta, Miroslav and Pivoňková, Hana},
article_location = {HEIDELBERG},
article_number = {24},
doi = {http://dx.doi.org/10.1007/s00216-014-7996-0},
keywords = {Electrochemical analysis; Labeled probes; Osmium complex; Tumor suppressor protein p53; DNA-protein interaction; Immunoprecipitation; Competition assay; Magnetic beads; Mercury electrode},
language = {eng},
issn = {1618-2642},
journal = {Analytical and Bioanalytical chemistry},
title = {Electrochemical detection of DNA binding by tumor suppressor p53 protein using osmium-labeled oligonucleotide probes and catalytic hydrogen evolution at the mercury electrode},
url = {http://download.springer.com/static/pdf/605/art%253A10.1007%252Fs00216-014-7996-0.pdf?auth66=1424693573_e49fe0b8548f527d28393c19da86150b&ext=.pdf},
volume = {406},
year = {2014}
}
TY - JOUR
ID - 1227600
AU - Němcová, Kateřina - Šebest, Peter - Havran, Luděk - Orság, Petr - Fojta, Miroslav - Pivoňková, Hana
PY - 2014
TI - Electrochemical detection of DNA binding by tumor suppressor p53 protein using osmium-labeled oligonucleotide probes and catalytic hydrogen evolution at the mercury electrode
JF - Analytical and Bioanalytical chemistry
VL - 406
IS - 24
SP - 5843-5852
EP - 5843-5852
PB - SPRINGER HEIDELBERG
SN - 16182642
KW - Electrochemical analysis
KW - Labeled probes
KW - Osmium complex
KW - Tumor suppressor protein p53
KW - DNA-protein interaction
KW - Immunoprecipitation
KW - Competition assay
KW - Magnetic beads
KW - Mercury electrode
UR - http://download.springer.com/static/pdf/605/art%253A10.1007%252Fs00216-014-7996-0.pdf?auth66=1424693573_e49fe0b8548f527d28393c19da86150b&ext=.pdf
L2 - http://download.springer.com/static/pdf/605/art%253A10.1007%252Fs00216-014-7996-0.pdf?auth66=1424693573_e49fe0b8548f527d28393c19da86150b&ext=.pdf
N2 - In this paper, we present an electrochemical DNA-protein interaction assay based on a combination of protein-specific immunoprecipitation at magnetic beads (MBIP) with application of oligonucleotide (ON) probes labeled with an electroactive oxoosmium complex (Os,bipy). We show that double-stranded ONs bearing a dT(20) tail labeled with Os,bipy are specifically recognized by the tumor suppressor p53 protein according to the presence or absence of a specific binding site (p53CON) in the double-stranded segment. We demonstrate the applicability of the Os,bipy-labeled probes in titration as well as competition MBIP assays to evaluate p53 relative affinity to various sequence-specific or structurally distinct unlabeled DNA substrates upon modulation of the p53-DNA binding by monoclonal antibodies used for the immunoprecipitation. To detect the p53-bound osmium-labeled probes, we took advantage of a catalytic peak yielded by Os,bipy-modified DNA at the mercury-based electrodes, allowing facile determination of subnanogram quantities of the labeled oligonucleotides. Versatility of the electrochemical MBIP technique and its general applicability in studies of any DNA-binding protein is discussed.
ER -
NĚMCOVÁ, Kateřina, Peter ŠEBEST, Luděk HAVRAN, Petr ORSÁG, Miroslav FOJTA a Hana PIVOŇKOVÁ. Electrochemical detection of DNA binding by tumor suppressor p53 protein using osmium-labeled oligonucleotide probes and catalytic hydrogen evolution at the mercury electrode. \textit{Analytical and Bioanalytical chemistry}. HEIDELBERG: SPRINGER HEIDELBERG, roč.~406, č.~24, s.~5843-5852. ISSN~1618-2642. doi:10.1007/s00216-014-7996-0. 2014.
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