title: [Application of two-color infrared fluorescence for detection of protein phosphorylation].
abstract: OBJECTIVE
  To assess the value of two-color infrared fluorescence imaging system in detecting protein phosphorylation in comparison with chemiluminescent detection.
  
  
  METHODS
  The lung tissue homogenate of mice treated with lipopolysaccharide (LPS) for different time lengths were prepared to separate the proteins by SDS-polyacrylamide gel electrophoresis followed by transfer of the proteins onto PVDF membranes. The membranes were incubated with the antibodies against total p42/44 MAPK/phospho-p42/44 MAPK, and then with goat anti-mouse or anti-rabbit secondary antibodies conjugated to Alexa Fluor 680, IRDye 800 or horseradish peroxidase. The blotted proteins were detected and quantified using Odyssey infrared imaging system or chemiluminescent detection.
  
  
  RESULTS
  LPS treatment rapidly induced p42/44 MAPK phosphorylation, which reached the peak level 1 h after the treatment and resumed the baseline level at 12 h. Consistent results were obtained by the two detection methods, but two-color infrared fluorescence imaging system showed better sensitivity in detecting the target protein, and was easy to manipulate with good efficiency and capable of analyzing two proteins simultaneously.
  
  
  CONCLUSIONS
  Two-color infrared fluorescence system is a powerful system for detecting phosphorylation of proteins.
authors.affiliation: Department of Pathophysiology and Key Laboratory of Functional Proteomics of Guangdong Province, Southern Medical University, Guangzhou 510515, China. liujhua@fimmu.com
authors.forename: Jing-hua
authors.initials: JH
authors.lastname: Liu
authors.forename: Jing
authors.initials: J
authors.lastname: Tang
authors.forename: Xing-guo
authors.initials: XG
authors.lastname: Lan
authors.forename: Ya-wei
authors.initials: YW
authors.lastname: Liu
authors.forename: Zhi-jie
authors.initials: ZJ
authors.lastname: Li
authors.forename: Fang
authors.initials: F
authors.lastname: Cheng
authors.forename: Peng
authors.initials: P
authors.lastname: Deng
authors.forename: Yong
authors.initials: Y
authors.lastname: Jiang
mesh_terms: D000818:Animals; D015153:Blotting, Western / Q000295:instrumentation / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D008070:Lipopolysaccharides; D008163:Luminescent Measurements / Q000295:instrumentation / Q000379:methods; D008168:Lung / Q000187:drug effects / Q000201:enzymology / Q000473:pathology; D051379:Mice; D008807:Mice, Inbred BALB C; D019950:Mitogen-Activated Protein Kinase 1 / Q000737:chemistry / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D015203:Reproducibility of Results; D012772:Shock, Septic / Q000139:chemically induced / Q000201:enzymology
chemical_list: D005456:Fluorescent Dyes; D008070:Lipopolysaccharides; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3