The objective of Phase I is to prepare a new family of chemiluminescent label compounds which can be conjugated to proteins and nucleic acids, subjected to electrophoresis and triggered to generate chemiluminescence in the gel. Successful development of the labeling compounds will permit the development of simple methods for direct detection in gels by eliminating membrane transfer and immunological binding protocols used in many techniques. The patented chemiluminescent technology involves reaction of the compounds with inexpensive chemical reagents. A prototypical compound produces visible chemiluminescence within an electrophoresis gel when conjugated to a protein. Additional chemiluminescent compounds emitting at distinct wavelengths spanning the visible spectrum will be synthesized and tested. Synthetic methods for conjugation will be established. Linkers for coupling to amine and thiol groups and a phosphoramidite linker are targeted. The chemiluminescence intensity, kinetics and spectrum of the chemiluminescent compounds will be evaluated. A key feature of the chemiluminescent reaction is that light is emitted as a 1-2 second burst when triggered in solution so that light intensity will be as high as possible when triggering is conducted within a gel. Detection sensitivity in solution and gel matrices will then be assessed using labeled analytes in model assays. PROPOSED COMMERCIAL APPLICATION: The proposed research seeks to develop a family of chemiluminescent direct labels which will enable the rapid and sensitive separation and detection of labeled protein and nucleic acid analytes within electrophoresis gels. The new method would greatly simplify analyses which now rely on membrane transfer or immunological binding protocols. Envisioned uses include preparing labeled polypeptide and nucleic acid size markers, 2D protein analysis, electrophoretic mobility shift analysis of transcriptional factors, nucleic acid hybridization assays and analysis of gene expression levels.
Thesaurus Terms: affinity labeling, chemical conjugate, chemical synthesis, gel electrophoresis, luminescence, nucleic acid quantitation /detection, technology /technique development chemical kinetics
