RAD-FREE®
Non-Isotopic Labeling and Detection System

This proven, highly sensitive, non-radioactive labeling and detection method has a number of superior features that are convincing enough to cause a switch away from radioactive tags.

• Real, and consistent detection levels in the low femtogram range - equivalent to high specific-activity 32P-labeled methods
• Rapid interaction of psoralin with nucleic acid
• Label both single and double-stranded nucleic acids
• Rapid and efficient UV cross-linking of label.
• Label probes in solution or in gels
• Store labeled probes for months or more without loss of activity
• Use RAD-FREE probes for detection in Southern and Northern blots

The RAD-FREE labeling and detection system is a 3 step process:

1. The first step involves binding biotin to the label target: Psoralens intercalate into nucleic acids and are covalently linked after UV exposure. The RAD-FREE System uses a psoralen - biotin com- plex, where psoralen is bound to biotin via a 14 atom linker, that has a high affinity for nucleic acids. Because this psoralen - biotin complex will not bind to proteins or other contaminants, sample preparation has little effect on labeling. Intercalation occurs in low salt solutions, <20 mM, and at pHs from 2.5 to 10. Enzymatic labeling methods, on the other hand, are often affected by pH, salt concentrations, and the presence of proteins or other molecules present with the target.



2. The second step uses streptavidin/biotin interaction to localize alkaline phosphatase on the target molecule. To minimize aggre- gates, the RAD-FREE enzyme complex is conjugated with hetero- bifunctional reagents and size-selected for an equal ratio of enzyme to streptavidin.



3. The third step places a highly sensitive chemiluminescent sub- strate, Lumingenš PPD, in contact with the alkaline phosphatase where it is dephosphorylated. This forms an unstable intermediate that decays and emits light in proportion to the concentration of enzyme. The RAD-FREE System uses a simple method of present- ing the substrate to the enzyme. The blot that has been hybridized to the labeled probe is laid on a substrate-impregnated sheet. The substrate migrates into the blot allowing the reaction to occur. Because substrate migration is continuous over several hours to days, film exposure can be regulated with minimal time concerns.