QUANTITATIVE DOT-BLOT ASSAY FOR PROTEINS USING ENHANCED CHEMILUMINESCENCE

A sensitive non-radioactive method for detection of specific proteins on Western blots is commercially available. The protein is immobilized on nitrocellulose membrane and immunolabelled with HRP-conjugated secondary antibody. HRP catalyzes the oxidation of luminol, a cyclic diacylhydrazide, resulting in the emission of light which is recorded on film. Using dot blot, we have shown that the signal generated by this system is proportional to the amount of protein loaded onto the membrane. Standard curves were linear (r2 > 0.95) over a 10-50-fold range. Linearity was also achieved with tissue extracts probed for a specific antigen. The sensitivity of the method is such that less than 10 fmol protein can be measured. The sensitivity and range are comparable to a previously reported dot-blotting assay based on the use of I-125-protein A, but the method does not require the handling of radioactive compounds. This method was used to estimate the level of chromogranin A in a mixture of proteins extracted from human brain.