Detection of c-Type Cytochromes Using Enhanced Chemiluminescence

✍ Scribed by C. Vargas; A.G. Mcewan; J.A. Downie

• Publisher: Elsevier Science
• Year: 1993
• Tongue: English
• Weight: 361 KB
• Volume: 209
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1993.1127

✦ Synopsis

A rapid, nontoxic method was devised for staining (c-) type cytochromes on filters. Bacterial and mitochondrial c-type cytochromes from sodium dodecyl sulfatepolyacrylamide 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^{\prime}, 5,5^{\prime})-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 \mathrm{~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. 1993 Academic Press, Inc.