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Effects of Various Additives on Cancer Biomarker Aptamer-Magnetic Pull-Down in Human Serum

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The effects of EDTA, dextran sulfate, and various detergents on DNA aptamer-conjugated magnetic bead pull-down of recombinant human ERK2 protein in buffer and human serum were investigated. Coomassie blue and silver-stained polyacrylamide gels revealed that a 192-base aptamer which was selected against a 19-amino acid peptide region of the human ERK2 protein in 25% trypsinized human plasma pulled down intact and fragmented ERK2 protein in phosphate buffered saline lacking calcium, but not in pure human serum. Addition of 2 mM EDTA enabled aptamer pulldown of ERK2 in serum. The identity of the recombinant ERK2 protein spiked into human serum samples and pulled down by aptamer-coated MBs was validated by mass spectrometry. Three unrelated aptamer sequences attached to MBs failed to pull down recombinant ERK2 in buffer. Electrophoretic banding pattern differences between various derivatized magnetic beads following exposure to human serum and acid elution were also examined in the presence and absence of dextran sulfate and various detergents to determine the best method for reducing nonspecific binding. Data suggest that additives can improve the binding affinity and specificity or purity of aptamer magnetic pull-down assays, but may also diminish target capture efficiency. Therefore, the specific effects of each additive on particular prototypical aptamer pull-down assays should be carefully studied and characterized by electrophoresis prior to use in more sophisticated mass spectral or other analytical techniques.
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Keywords: APTAMER; CALCIUM; ERK2; MAGNETIC BEAD; PULL-DOWN ASSAY

Document Type: Research Article

Publication date: February 1, 2017

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  • Bionanoscience attempts to harness various functions of biological macromolecules and integrate them with engineering for technological applications. It is based on a bottom-up approach and encompasses structural biology, biomacromolecular engineering, material science, and engineering, extending the horizon of material science. The journal aims at publication of (i) Letters (ii) Reviews (3) Concepts (4) Rapid communications (5) Research papers (6) Book reviews (7) Conference announcements in the interface between chemistry, physics, biology, material science, and technology. The use of biological macromolecules as sensors, biomaterials, information storage devices, biomolecular arrays, molecular machines is significantly increasing. The traditional disciplines of chemistry, physics, and biology are overlapping and coalescing with nanoscale science and technology. Currently research in this area is scattered in different journals and this journal seeks to bring them under a single umbrella to ensure highest quality peer-reviewed research for rapid dissemination in areas that are in the forefront of science and technology which is witnessing phenomenal and accelerated growth.
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