Plant & Animal Genome IX Conference

Workshop: LICOR - Automated Genetic Analysis
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INFRARED FLUORESCENT LABELING TECHNOLOGY FOR THE ANALYSIS OF MEMBRANE ARRAYS AND SOUTHERN, NORTHERN, AND WESTERN BLOTS

LICOR, Inc., 4308 Progressive Ave., Lincoln, NE 68504

The use of fluorescent 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 were directly labeled with carbodiimide derivatized IR dyes in a 10 minute labeling and clean up procedure. IR labeled DNA probes could detect as little as 128 zeptomoles of DNA in a Southern blot format, a level of sensitivity equivalent to that reported for chemiluminescent detection. An IR dye labeled cDNA derived from YP2 mRNA of drosophila detected the homologous mRNA in 0.55 micrograms of total RNA, a level of sensitivity equivalent to that reported for radioisotopic detection. IR labeled human prostate cDNA detected prostate specific gene expression when hybridized against a membrane array of human cDNAs. IR labeled secondary antibodies detected a variety of proteins in Western blots with sensitivity equivalent to chemiluminescent detection methods without the additional labor and cost of the chemiluminescent substrates. In addition, the IR dye labeled antibodies enabled simultaneous multicolor analysis of multiple proteins, a technique not possible with chemiluminescence methods. IR dye methods should provide increased sensitivity, speed, simplicity, and multicolor capability for membrane based analysis of proteins and nucleic acids.