[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.
Method: 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.
Conclusion: Two-color infrared fluorescence system is a powerful system for detecting phosphorylation of proteins.