Purification of the Ah receptor from C57BL/6J mouse liver.
The photoaffinity ligand for the Ah receptor, [125I]-2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin, previously has been shown to selectively label two peptides in the cytosol fraction of C57BL/6J mouse liver: a 95-kDa peptide, the ligand binding moiety of the Ah receptor, and a 70-kDa proteolytic fragment formed from the larger peptide (Poland, A., Glover, E., Ebetino, F. H., and Kende, A.S. (1986) J. Biol. Chem. 261, 6352-6365). These two peptides were partially purified to an approximately 20,000-fold enrichment with a 15-20% yield by the following scheme: 1) photoaffinity labeling of the 35-55% ammonium sulfate fraction of liver cytosol; 2) chromatography on polyethyleneimine-Sepharose coupled at low charge density and heparin/Mn2+ precipitation of the dilute column eluate; 3) DEAE-Sepharose chromatography to remove heparin; 4) chromatography on heparin-Sepharose; 5) preparative sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis followed by electroelution of the protein and ion pair extraction to remove sodium dodecyl sulfate; and 6) high performance liquid chromatography on a reverse-phase C-4 column. Following initial chromatography on polyethyleneimine Sepharose, it was found that substantial subsequent purification could only be achieved under denaturing conditions.
Perdew
G H
GH
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Poland
A
A
D000345:Affinity Labels; D000645:Ammonium Sulfate; D000818:Animals; D011232:Chemical Precipitation; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000032:analysis; D004147:Dioxins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000032:analysis*; D051379:Mice; D008810:Mice, Inbred C57BL; D010777:Photochemistry; D011489:Protein Denaturation; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug / Q000302:isolation & purification* / Q000378:metabolism
D000345:Affinity Labels; D004147:Dioxins; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug; C054916:2-azido-3-iodo-7,8-dibromodibenzo-1,4-dioxin; D000645:Ammonium Sulfate