[Application of two-color infrared fluorescence for detection of protein phosphorylation].
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.
Liu
Jing-hua
JH
Department of Pathophysiology and Key Laboratory of Functional Proteomics of Guangdong Province, Southern Medical University, Guangzhou 510515, China. liujhua@fimmu.com
Tang
Jing
J
Lan
Xing-guo
XG
Liu
Ya-wei
YW
Li
Zhi-jie
ZJ
Cheng
Fang
F
Deng
Peng
P
Jiang
Yong
Y
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
D005456:Fluorescent Dyes; D008070:Lipopolysaccharides; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3