Rapid detection of two-protein interaction with a single fluorophore by using a microfluidic device.
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).
Chou
Chao-Kai
CK
Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA.
Jing
Nan
N
Yamaguchi
Hirohito
H
Tsou
Pei-Hsiang
PH
Lee
Heng-Huan
HH
Chen
Chun-Te
CT
Wang
Ying-Nai
YN
Hong
Sungmin
S
Su
Chin
C
Kameoka
Jun
J
Hung
Mien-Chie
MC
D004815:Epidermal Growth Factor / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation; D011485:Protein Binding; D045663:Quantum Dots; D050796:STAT3 Transcription Factor / Q000032:analysis*; D019061:src-Family Kinases / Q000032:analysis* / Q000378:metabolism
D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D004815:Epidermal Growth Factor; D019061:src-Family Kinases