A novel probe for chemiluminescent image detection of proteins in two-dimensional gel electrophoresis

Carrier ampholytes were found to enhance the chemiluminescence (CL) emission from the 3-aminophthalic hydrazide (luminol)-hydrogen peroxide system. They can be used as a chemiluminescent probe for rapid detection of major proteins in gels. This probe attracted much interest due to its ability to attach proteins, and to the possibility to combine it with separation techniques generating the CL emission directly. Increased signal intensity was achieved employing optimized concentrations of the carrier ampholyte enhancer. The binding of carrier ampholyte to proteins was found to occur at the pI of the proteins. Proteins from different regions of the gels were identified by their matrix-assisted TOF mass spectra and by appropriate database search, the results illustrating the possibility of major protein detection in human serum. Direct CL image detection with the carrier ampholyte probe can be applied for the detection of characteristic proteins in patients, i.e., proteins which cannot be detected without the probe.