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

N. Padhan, J. Yan, A. Boge, E. Scrivener, H. Birgisson, A. Zieba, M. Gullberg, M. Kamali-Moghaddam, L. Claesson-Welsh, U. Landegren

Research output: Contribution to journalArticleAcademicpeer-review

11 Citations (Scopus)
91 Downloads (Pure)

Abstract

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.

Original languageEnglish
Article number1490
Number of pages9
JournalScientific Reports
Volume7
Issue number1
DOIs
Publication statusPublished - 4 May 2017

Keywords

  • Colorectal Neoplasms/diagnosis
  • Electrophoresis, Capillary/methods
  • Extracellular Signal-Regulated MAP Kinases/metabolism
  • Human Umbilical Vein Endothelial Cells/metabolism
  • Humans
  • Isoelectric Focusing/methods
  • Nanoparticles/chemistry
  • Nucleic Acid Amplification Techniques/methods
  • Phosphorylation
  • Proteins/analysis
  • Sensitivity and Specificity
  • Signal Transduction/drug effects
  • Vascular Endothelial Growth Factor A/pharmacology

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