A Universal Approach to Prepare Reagents for DNA-Assisted Protein Analysis
2014 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 9, no 9, e108061- p.Article in journal (Refereed) Published
The quality of DNA-labeled affinity probes is critical in DNA-assisted protein analyses, such as proximity ligation and extension assays, immuno-PCR, and immuno-rolling circle amplification reactions. Efficient, high-performance methods are therefore required for isolation of pure conjugates from reactions where DNA strands have been coupled to antibodies or recombinant affinity reagents. Here we describe a universal, scalable approach for preparing high-quality oligonucleotide-protein conjugates by sequentially removing any unconjugated affinity reagents and remaining free oligonucleotides from conjugation reactions. We applied the approach to generate high-quality probes using either antibodies or recombinant affinity reagents. The purified high-grade probes were used in proximity ligation assays in solution and in situ, demonstrating both augmented assay sensitivity and improved signal-to-noise ratios.
Place, publisher, year, edition, pages
2014. Vol. 9, no 9, e108061- p.
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
IdentifiersURN: urn:nbn:se:uu:diva-236454DOI: 10.1371/journal.pone.0108061ISI: 000342921200076PubMedID: 25233463OAI: oai:DiVA.org:uu-236454DiVA: diva2:764589