Phosphoprotein Detection by High-Throughput Flow Cytometry

Methods Mol Biol. 2016:1355:275-90. doi: 10.1007/978-1-4939-3049-4_19.

Abstract

Phospho flow cytometry is a powerful technique for the detection of protein phosphorylation events that, like Western blotting, relies on phospho-epitope-specific antibodies. In contrast to the latter, however, multidimensional and directly quantifiable data is obtained at the single-cell level allowing separate analysis of small cell populations in complex cellular mixtures. Furthermore, up to 30 phospho-specific antibodies or antibodies identifying other posttranslational modifications in combination with cell surface markers can be analyzed in a single experiment. Utilizing a technique called fluorescent cell barcoding that enables combination of up to 64 samples into one tube for multiplex analysis and later data deconvolution, phospho flow cytometry is turned into a medium- to high-throughput technology.

Keywords: Flow cytometry; Fluorescent cell barcoding; Phospho flow; Phospho-specific antibody; Signaling.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies / immunology
  • Antibody Specificity
  • Epitopes
  • Flow Cytometry*
  • Fluorescent Dyes / chemistry
  • High-Throughput Screening Assays*
  • Humans
  • Luminescent Measurements
  • Phosphoproteins / analysis*
  • Phosphoproteins / chemistry
  • Phosphoproteins / immunology
  • Phosphoproteins / metabolism
  • Phosphorylation
  • Protein Processing, Post-Translational
  • Proteomics / methods*
  • Workflow

Substances

  • Antibodies
  • Epitopes
  • Fluorescent Dyes
  • Phosphoproteins