Luminescent visualization of low amounts of cytochrome P450 and hemoproteins by luminol in acrylamide gels.
In this paper we apply luminol (5-amino-2,3-dihydro-1,4-phthalazinedione), a light-emitting substrate, in conjunction with H2O2 to the luminescence labeling of hemoproteins. We describe in detail a photodetection device which permits an efficient recording of the light emitted by heme-containing proteins resolved in acrylamide gels. The sensitivity of this procedure, when compared to the classical 3,3'-5,5'-tetramethylbenzidine staining method, results in a 5- to 20-fold enhancement with standard hemoproteins (lactoperoxidase, catalase, cytochrome P450 2B4, horseradish peroxidase, hemoglobin, myoglobin, and cytochrome b5). The potential applications of this technique are illustrated by the detection of cytochrome P450 in microsomes from plant as well as from animal extrahepatic tissues which possess low amounts of this cytochrome.
Bonfils
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C
INSERM Unité 128, CNRS, Montpellier, France.
Charasse
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S
Bonfils
J P
JP
Larroque
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C
D000818:Animals; D001923:Brain Chemistry; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis*; D015786:Cytochromes b5 / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation / Q000379:methods; D006420:Hemeproteins / Q000032:analysis*; D006454:Hemoglobins / Q000032:analysis; D008099:Liver / Q000737:chemistry; D008163:Luminescent Measurements; D008165:Luminol; D008861:Microsomes / Q000737:chemistry; D009211:Myoglobin / Q000032:analysis; D010544:Peroxidases / Q000032:analysis*; D010944:Plants / Q000737:chemistry; D051381:Rats; D012680:Sensitivity and Specificity
D006420:Hemeproteins; D006454:Hemoglobins; D009211:Myoglobin; D008165:Luminol; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D010544:Peroxidases