Rapid detection of two-protein interaction with a single fluorophore by using a microfluidic device

Analyst. 2010 Nov;135(11):2907-12. doi: 10.1039/c0an00229a. Epub 2010 Sep 28.

Abstract

We have developed a microfluidics based platform and methodology named MAPS (microfluidic system for analyzing proteins in single complex) for detecting two protein interactions rapidly using a single fluorophore. Target proteins were labelled with Quantum dot 525 (QD525) via specific polyclonal antibodies, and were transported through the microfluidic channel subsequently, where the 375 nm excitation laser light was focused to form a detection volume. Photon bursts from target proteins passing through the detection volume were recorded and their photon burst histograms were plotted which demonstrated roughly the specific protein interaction ratio based on their population and statistical behavior. As a proof of concept, Src/STAT3 protein complex interaction ratios with and without EGF stimulation were obtained by MAPS within 1 h and the results were well matched with the one obtained by the conventional immunoprecipitation/Western blot (IP/WB).

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Epidermal Growth Factor / chemistry
  • Fluorescent Dyes / chemistry*
  • HeLa Cells
  • Humans
  • Immunoglobulin G / chemistry
  • Microfluidic Analytical Techniques* / instrumentation
  • Protein Binding
  • Quantum Dots
  • STAT3 Transcription Factor / analysis*
  • src-Family Kinases / analysis*
  • src-Family Kinases / metabolism

Substances

  • Fluorescent Dyes
  • Immunoglobulin G
  • STAT3 Transcription Factor
  • STAT3 protein, human
  • Epidermal Growth Factor
  • src-Family Kinases