Electrotransfer of microsomal cytochrome P450 after isoelectric focusing (IEF blots): resolution of human 2A and 3A isozymes.
In this article a procedure is described which allows us to achieve electroblots of membrane-associated proteins after isoelectric focusing. Proteins are resolved in vertical acrylamide slab gels containing ampholines, urea, and nonionic detergents (dodecyl maltoside and Tergitol NP10). After migration the focusing gel is reinforced by a carrier gel (10% acrylamide) polymerized on only one face of the slab. Electroelution of the proteins toward a hydrophobic membrane (Immobilon P) is obtained by adding denaturing agents to the carrier gel (sodium dodecyl sulfate and mercaptoethanol) and by increasing the methanol concentration (60%) in the transfer buffer. This method is applied to analyzing P450 2A and 3A cytochromes from human liver microsomes.
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Institut National de la Santé et de la Recherche Médicale, Unité 128, BP 5051, Montpellier, France.
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D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004579:Electron Transport; D006801:Humans; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000378:metabolism*; D008099:Liver / Q000166:cytology / Q000201:enzymology*; D008565:Membrane Proteins / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology*; D012995:Solubility
D007527:Isoenzymes; D008565:Membrane Proteins; D003577:Cytochrome P-450 Enzyme System