Enhancement of diaminobenzidine colorimetric signal in immunoblotting.

Immunodetection methods such as immunohistochemistry and immunoblotting (Western blot analysis) are used to specifically and sensitively detect proteins of interest. In both immunohistochemical and immunoblotting techniques, a primary antibody binds to the protein of interest, and through the subsequent detection of this immunoglobulin with a conjugated secondary antibody, the presence of the protein of interest can be visualized using radioisotopic or enzymatic techniques. We report that the combination of 3,3′-diaminobenzidine (DAB) with imidazole and metal enhancement is a highly sensitive visualization technique for Western blot analysis. This procedure gives an intense brownor blue/grey-colored reaction product, a high signal-to-noise ratio and greatly enhanced product detection compared to the standard DAB protocol. Many visualization protocols use secondary antibodies conjugated directly to enzymes or linked to amplification systems that use various substrates to produce a detectable product. Enzymes most frequently used for these procedures include alkaline phosphatase and horseradish peroxidase (HRP). Studies have found that for light and electron immunohistochemistry, DAB is an exceptionally good substrate for HRP because, upon oxidation, DAB polymerizes to form an intense browncolored precipitate that is insoluble in aqueous or organic solvents (3). Further studies have shown that for immunohistochemical staining, both the sensitivity and intensity of the standard HRP/DAB protocol of Graham and Karnovsky (3) can be increased by acidifying the buffer closer to the optimum pH for HRP enzymatic activity (13,14) or by the addition of imidazole to the reaction buffer (10,11,14). HRP catalyzes the transfer of electrons from DAB, resulting in its oxidation. The addition of imidazole has been suggested to induce formation of a third electron transfer site in HRP, thereby increasing its activity (10). Imidazole has been reported to increase HRP oxidation of DAB approximately fivefold (12). Finally, the addition of metals such as nickel, cobalt, copper and silver has been shown to improve DAB polymer formation and can change the color of the final reaction product (1,5,8). The nature of the DAB-metallic ion interaction is not definitively known but may be similar to the deposition of osmium in DAB polymers (5,9). Immunoblotting, or Western blotting, is used to detect the amounts of specific protein in cell or tissue extracts after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretic transfer of the proteins onto a nitrocellulose or polyvinylidene difluoride (PVDF) filter (2,4). Following binding of a primary antibody and secondary antibody, both colorimetric and chemiluminescencebased detection of HRP or alkaline phosphatase are commonly used to generate a detectable signal. Chemiluminescence-based procedures are generally considered more sensitive than colorimetric detection (7). In this study, Western blot analysis was performed, and the sensitivity and intensity of reaction product after visualization using the standard HRP/DAB oxidation protocol (3), with and without metals, was compared with imidazole-enhanced HRP/DAB with and without metals. Other HRP substrates (aminoethylcarbazole) or other intensification procedures (lower pH, silver or copper enhancement) were also tested and found to give a much lower signal-