A Universal Approach to Prepare Reagents for DNA-Assisted Protein Analysis |
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Authors: | Junhong Yan Gucci Jijuan Gu Christian Jost Maria Hammond Andreas Plückthun Ulf Landegren Masood Kamali-Moghaddam |
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Institution: | 1. Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.; 2. Department of Genetics, Stanford University, Palo Alto, California, United States of America.; 3. Department of Biochemistry, University of Zurich, Zurich, Switzerland.; Deutsches Krebsforschungszentrum, Germany, |
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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. |
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