Purification of the Ah receptor from C57BL/6J mouse liver

G. H. Perdew, A. Poland

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Abstract

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 ~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 electrodilution 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.

Original languageEnglish (US)
Pages (from-to)9848-9852
Number of pages5
JournalJournal of Biological Chemistry
Volume263
Issue number20
StatePublished - 1988

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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