A universal approach to prepare reagents for DNA-assisted protein analysis.

Yan J, Gu GJ, Jost C, Hammond M, Pl├╝ckthun 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.

Affiliated researcher

PubMed 25233463

DOI 10.1371/journal.pone.0108061

Crossref 10.1371/journal.pone.0108061

pii: PONE-D-14-28123
pmc: PMC4169473