The application of infrared fluorescent technology for the analysis of proteins and nucleic acids

The use of fluorescently labeled probes for membrane-based analysis of proteins and nucleic acids have been unsuccessful due to high background fluorescence of the membranes. We have developed a system for the analysis of proteins and nucleic acids using near infrared (IR) fluorescent dyes and a new scanning instrument, the Odyssey Infrared Imager. Nucleic acid probes can be directly labeled with carbodiimide-derivatized IR dyes in a 10 minute labeling and clean up procedure. In a Northern blot format, mRNA from genes such as mouse beta-actin, mouse GAPDH, and cyclophilin mRNA have been detected in as little as 0.1 mug of total RNA. In immunoblotting assays, IR detection of proteins was as sensitive as chemiluminescence and routinely enabled detection of 1-10 pg of protein. Signal transduction events in the MAP kinase pathway were analyzed using two-color immunoblotting. Phosphorylated and non-phosphorylated proteins were detected and quantitated simultaneously without the need for stripping and reprobing. A model system for assessing protein-nucleic acid interactions by EMSA was developed based on the binding of T7 RNA polymerase to its promoter. Varying amounts of protein were combined with a PCR-generated IRD800-labeled DNA fragment. Protein binding could be monitored quantitatively following scanning of the gel on the Odyssey Infrared Imager. The Odyssey Infrared Imaging system permits two-color analysis of proteins and nucleic acids with very high signal to noise ratios.