title: A universal approach to prepare reagents for DNA-assisted protein analysis.
abstract: 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.
authors.affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Junhong
authors.initials: J
authors.lastname: Yan
authors.affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden; Department of Genetics, Stanford University, Palo Alto, California, United States of America.
authors.forename: Gucci Jijuan
authors.initials: GJ
authors.lastname: Gu
authors.affiliation: Department of Biochemistry, University of Zurich, Zurich, Switzerland.
authors.forename: Christian
authors.initials: C
authors.lastname: Jost
authors.affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Maria
authors.initials: M
authors.lastname: Hammond
authors.affiliation: Department of Biochemistry, University of Zurich, Zurich, Switzerland.
authors.forename: Andreas
authors.initials: A
authors.lastname: Plückthun
authors.affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Ulf
authors.initials: U
authors.lastname: Landegren
authors.affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Masood
authors.initials: M
authors.lastname: Kamali-Moghaddam
mesh_terms: D045744:Cell Line, Tumor; D015342:DNA Probes / Q000235:genetics; D006801:Humans; D015336:Molecular Probe Techniques; D021141:Nucleic Acid Amplification Techniques; D020543:Proteome / Q000378:metabolism*; D059629:Signal-To-Noise Ratio
chemical_list: D015342:DNA Probes; D020543:Proteome