IMAGING OF CHEMILUMINESCENT SIGNALS WITH COOLED CCD CAMERA SYSTEMS

We investigated imaging of chemiluminescent signals from 1,2-dioxetane s with cooled CCD cameras. Non-radioactive detection methods for biomo lecules utilizing these chemiluminescent substrates for alkaline phosp hatase have been developed. Applications which have been successfully adapted to this technology include Southern and Northern blotting, imm unoblotting, ELISA methods and DNA sequencing. Dephosphorylation of th e dioxetane CSPD by alkaline phosphatase generates an unstable anion t hat decomposes resulting in light production. The wavelength of the em itted light is approximately 460 nm. We have utilized Photometrics Sta r and MXC 200L cooled CCD cameras for direct imaging of chemiluminesce nt signals. Benefits of utilizing a CCD detector include rapid data di gitization and more accurate quantitation of chemiluminescent signals compared to film-based densitometry owing to the significantly greater dynamic range. Chemiluminescent images from dot blots of biotinylated DNA, Southern blots and DNA sequencing gel blots were obtained. In a chemiluminescent microtitre plate assay, serial dilutions of alkaline phosphatase spanning four orders of magnitude can be detected. Our res ults indicate that the digitization of chemiluminescent signal data wi th cooled CCD cameras is an excellent alternative to P-32 detection me thods utilizing storage phosphor screen imaging systems.