USE OF DNA OLIGONUCLEOTIDES AND SSDNA APTAMERS AS PROTEIN DETECTION TOOLS COMPLEMENTARY TO WESTERN BLOTS

CRISTIAN JORGE ALEJANDRO ASENSIO; T. A. SANTA COLOMA

Mar del Plata

Congreso; LXI REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INVESTIGACIÓN CLÍNICA (SAIC); 2016

SAIC

The use of DNA as reagent in the detection of proteins bound to nitrocellulose or other membranes is known as south-western technique and it is equivalent to the much better known western-blot technique that employs antibodies. In here, we proposed to determine if the use of biotinylated DNA oligonucleotides can be a tool complementary (or supplementary) of western blots. We explored the profiles produced by the binding of different oligonucleotides to proteins resolved by SDS-PAGE and present in lysates of different cell lines or organs. We determined the way the oligonucleotides interact with the different proteins including histones. Interestingly, by modulating the composition of the solutions employed for membrane blocking, DNA binding and washing we were able to control which one of the histones is detected preferentially. On the other hand, when the aim was to avoid histone detection, we were also able to prevent it by adding some components to the binding and/or the blocking buffers. Furthermore, we studied the compatibility of the use of oligonucleotides in membranes already employed with antibodies, stripped or not. Besides, we determined the capacity of the oligonucleotides to bind histones and other proteins in the presence of salts and detergents together with the optimal solution for oligonucleotide stripping and membrane re-probing. Finally, to evaluate the usefulness of our technique characterizations in the field of DNA aptamers, we tested a ssDNA aptamer sequence previously published as specific for a human core histone peptide but that had not been selected or validated in the context of a cell lysate or of the other histones. Interestingly, this aptamer was not able to differentiate its intended histone target from the other core histones present in the lysates. We hope that our findings and strategies will improve DNA aptamer selection programs in general as well as to facilitate studies of histones and other proteins in blotting membranes