EVALUATING APTABLOTS AND SOUTHWESTERN- BLOT ASSAYS AS ALTERNATIVES FOR THE SENSITIVE DETECTION OF ALL HISTONES IN TOTAL CELL LYSATES

CRISTIAN JORGE ALEJANDRO ASENSIO; M. MASSIP COPIZ; M. CLAUZURE; T. A. SANTA COLOMA

BUENOS AIRES

Congreso; LXII REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INVESTIGACIÓN CLÍNICA (SAIC); 2017

SAIC

Abstract: Histones PTMs are markers of epigenetic events andextracellular histones modulate inflammatory, autoimmune and innateimmune responses. It is useful to detect histones by sensitiveassays. Since they evolved to bind DNA, several biotinylated oligonucleotidesand one aptamer were evaluated as reagents for thecolorimetric or chemiluminescent detection of endogenous histonesbound to nitrocellulose. Many assay variables were systematicallyadjusted. These south-western or aptablot techniques resultedsuperior alternatives to western-blot with antibodies. Cell lines ororgans were lysed to study the oligo-histones binding profiles resolvedby SDS-PAGE. For the TOTAL DETECTION of the 5 histones,biotinylated DNA was an optimal, cost-effective tool, complementaryor supplementary of antibodies. We characterized oligonucleotide-histones interactions modulating the components of solutionsin the steps of membrane blocking, DNA binding or washing andcontrolled which histone was detected preferentially adjusting theionic strength. When needed, histone detection was completely preventedby adding polyanionic reagents (p-value ≤0.01). Testing 1ssDNA aptamer published as H4 specific but not selected/validatedin a lysate context, it reproducibly failed (p-value ≤0.01) to differentiateits H4 target from the other core histones indicating that the selectionenvironment is important in specificity determination. Finally,we developed novel, reliable, non-proteic, coloured MW trackers forbetter histone resolution in gels. Our procedures showed reproducibleanalytical advantages and may aid the design of histone bioassayplatforms. With a single reagent at picomolar concentrationall histones can be sensitively detected for signal normalization inmembranes. Our strategies are expected to improve DNA aptamerselection protocols and to facilitate studies of histones for epigenetic,proteomic or immunity research. Keywords: histones, epigenetic,proteomics