Highly sensitive and specific protein detection via combined capillary isoelectric focusing and proximity ligation

Padhan, Narendra (author): Uppsala universitet,Vaskulärbiologi,Science for Life Laboratory, SciLifeLab,Rudbeck Lab, S-75185 Uppsala, Sweden.

Yan, Junhong (author): Uppsala universitet,Institutionen för immunologi, genetik och patologi,Science for Life Laboratory, SciLifeLab,Rudbeck Lab, S-75185 Uppsala, Sweden.;Eindhoven Univ Technol, Inst Complex Mol Syst, Dept Biomed Engn, NL-5600 MB Eindhoven, Netherlands.

Boge, Annegret (author): Protein Simple, 3001 Orchard Pkwy, San Jose, CA 95134 USA.

Scrivener, Elaine (author): Protein Simple, 3001 Orchard Pkwy, San Jose, CA 95134 USA.

Zieba, Agata (author): Uppsala universitet,Institutionen för immunologi, genetik och patologi,Science for Life Laboratory, SciLifeLab,Rudbeck Lab, S-75185 Uppsala, Sweden.

Gullberg, Mats (author): Olink Biosci, Dag Hammarskjolds Vag 52B, S-75237 Uppsala, Sweden.

Kamali-Moghaddam, Masood (author): Uppsala universitet,Molekylära verktyg,Science for Life Laboratory, SciLifeLab,Rudbeck Lab, S-75185 Uppsala, Sweden.

Claesson-Welsh, Lena (author): Uppsala universitet,Vaskulärbiologi,Science for Life Laboratory, SciLifeLab,Rudbeck Lab, S-75185 Uppsala, Sweden.

Landegren, Ulf (author): Uppsala universitet,Molekylära verktyg,Science for Life Laboratory, SciLifeLab,Rudbeck Lab, S-75185 Uppsala, Sweden.

(creator_code:org_t)

Related links:: https://doi.org/10.1...; show more...; https://uu.diva-port... (primary) (Raw object); https://www.nature.c...; https://urn.kb.se/re...; https://doi.org/10.1...; show less...

Abstract Subject headings

Detection and quantification of proteins and their post-translational modifications are crucial to decipher functions of complex protein networks in cell biology and medicine. Capillary isoelectric focusing together with antibody-based detection can resolve and identify proteins and their isoforms with modest sample input. However, insufficient sensitivity prevents detection of proteins present at low concentrations and antibody cross-reactivity results in unspecific detection that cannot be distinguished from bona fide protein isoforms. By using DNA-conjugated antibodies enhanced signals can be obtained via rolling circle amplification (RCA). Both sensitivity and specificity can be greatly improved in assays dependent on target recognition by pairs of antibodies using in situ proximity ligation assays (PLA). Here we applied these DNA-assisted RCA techniques in capillary isoelectric focusing to resolve endogenous signaling transducers and isoforms along vascular endothelial growth factor (VEGF) signaling pathways at concentrations too low to be detected in standard assays. We also demonstrate background rejection and enhanced specificity when protein detection depended on binding by pairs of antibodies using in situ PLA, compared to assays where each antibody preparation was used on its own.

By the author/editor: Padhan, Narendra; Yan, Junhong; Boge, Annegret; Scrivener, Elain ...; Birgisson, Helgi; Zieba, Agata; show more...; Gullberg, Mats; Kamali-Moghaddam ...; Claesson-Welsh, ...; Landegren, Ulf; show less...

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