Detection of c-type cytochromes using enhanced chemiluminescence

A rapid, nontoxic method was devised for staining c-type cytochromes on filters. Bacterial and mitochondrial c-type cytochromes from sodium dodecyl sulfate-polyacrylamide gels were transferred to nitrocellulose filters and visualized by a staining system that relies on their heme peroxidase activity. This involved the use of enhanced chemiluminescence (ECL). This system was compared with the 3,3',5,5'-tetramethylbenzidine (TMBZ) staining of cytochromes transferred to nitrocellulose filters and found to be equally sensitive. The ECL assay has the following advantages: (i) the signals can be quantitated with a densitometer; (ii) it is much quicker than the TMBZ-staining protocol (20 min versus 2 h); (iii) the generated signals are detected on a film providing a convenient record; (iv) after removal of the reagents, filters can be reused for immunoassays; and (v) it uses nonhazardous components.