Immunochemical detection method after western blotting of cytochrome P-450 iso-enzyme

In this report a number of staining techniques on Western blots have been compared with respect to sensitivity, background staining, practical applicability and cost aspects. After electrophoresis of a rat microsomal liver sample followed by blotting, an incubation was performed of a primary antibody against a purified cytochrome P-450 fraction. Enzyme-labelled second antibodies with horseradish peroxidase, alkaline phosphatase and xanthine oxidase were used for detection. Both colorimetric and chemiluminescent methods were investigated. It appeared that chemiluminescence detection of horseradish peroxidase was the most sensitive method. followed by chemiluminescence detection of xanthine oxidase. Horseradish peroxidase with colorimetric detection was also fairly sensitive and easier to use than chemiluminescence detection. Alkaline phosphatase gave the least sensitive method with both colorimetric and chemiluminescence detection. Both horseradish peroxidase and especially alkaline phosphatase showed heavy background staining, in contrast to xanthine oxidase, where no background was observed even after overnight exposure. A number of blot experiments on nylonmembrane, for a more sensitive detection of alkaline phosphatase, did not give better results in sensitivity. Both colorimetric and chemiluminescence detection showed heavy background staining on nylonmembrane. The heavy background staining after chemiluminescence detection of horseradish peroxidase was less by using the second antibody in smaller concentration. The colorimetric detection of horseradish peroxidase gave better photographic reproduction when a substrate of diaminobenzidine and chloronaphthol was used for staining.RIV