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Yan J, Gu GJ, Jost C, Hammond M, Plückthun A, Landegren U, Kamali-Moghaddam M
PLoS ONE 9 (9) e108061 [2014-09-18; online 2014-09-18]
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.
Affinity Proteomics Uppsala [Technology development]
PLA and Single Cell Proteomics [Technology development]
PubMed 25233463
DOI 10.1371/journal.pone.0108061
Crossref 10.1371/journal.pone.0108061
pii: PONE-D-14-28123
pmc: PMC4169473