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.
John Innes Institute, John Innes Centre, Norwich, United Kingdom.
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C
Vargas
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McEwan
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JA
Downie
D000818:Animals; D001560:Benzidines; D002417:Cattle; D002863:Chromogenic Compounds; D003574:Cytochrome c Group / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D008163:Luminescent Measurements; D009206:Myocardium / Q000201:enzymology; D010544:Peroxidases / Q000737:chemistry; D011489:Protein Denaturation; D012967:Sodium Dodecyl Sulfate
D001560:Benzidines; D002863:Chromogenic Compounds; D003574:Cytochrome c Group; D012967:Sodium Dodecyl Sulfate; C021758:3,3',5,5'-tetramethylbenzidine; D010544:Peroxidases