Enhanced chemiluminescence peroxidase-conjugate

CYP3A4 protein expression can be measured directly by immunodetection (LeCluyse et ah, 2000 Luo et ah, 2002). Microsomal protein (3 jig) is resolved by SDS-polyacrylamide gel electrophoresis (12% acrylamide). Resolved proteins are transferred to nitrocellulose membranes, whieh are ineubated in 3% bovine serum albumin in phosphate-buffered saline supplemented with Tween 20 (0.1 M, pH 7.4, 0.1% Tween 20) for 45 min to block nonspecific protein binding. Membranes are then treated with primary antiCYP3A4 antibody (Gentest, Woburn, MA), followed by horseradish peroxidase-conjugated antimouse seeondary antibody. The antibody-reactive CYP3A4 protein bands are visualized using enhanced chemiluminescence detection and quantitated by photodensitometry (Desai et al., 2002). 

Total mouse liver proteins (50 were separated on a 10% sodium dodecylsulfate polyacrylamide gel (SDS-PAGE) and Western blotting was performed by electrophoretic transfer of the separated proteins onto a nitrocellulose filter (Nitropure, Micron Separations Inc., Westborough, MA, USA) using a Mini-Protean II device (Bio-Rad). The blots were probed with an anti-MTE-I antibody and subsequently with horseradish peroxidase-conjugated secondary antibodies and visuahsed by enhanced chemiluminescence (ECL, Amersham) using X-ray film. 

DNA probes can be labeled using the modified nucleotide, fluorescein-11-dUTP, and subsequently detected with enhanced chemiluminescence (1) using an antifluorescein antibody conjugated to horseradish peroxidase (2). The use of enhanced chemiluminescence avoids the fading of results associated with some colormetric detection procedures, used with nonradioactive labeling and detection systems. Hard copy results showing excellent resolution are produced. 

Develop the blots with horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence reagents (GE Healthcare, Piscataway, NJ). 

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