An assay for the detection of specific binding of 3-methylcholanthrene to rat liver cytosolic proteins using DEAE-cellulose |
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Authors: | B Tierney S Munzer E Bresnick |
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Affiliation: | Department of Biochemistry, Given Building, School of Medicine, University of Vermont, Burlington, Vermont 05405 USA |
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Abstract: | A method for the detection of the specific binding of 3-methylcholanthrene to rat liver cytosolic proteins is described. The separation of the protein-bound 3-methylcholanthrene from the free 3-methylcholanthrene was achieved using a batch DEAE-cellulose technique. Extraction of the DEAE-cellulose with 0.3 M KCl allowed the selective release and measurement of the amount of protein-bound 3-methylcholanthrene. The assay was optimized for the following parameters: time of incubation with DEAE-cellulose, time required for salt extraction, protein concentration, the concentration of KCl required to elute the specific binding proteins, the amount of DEAE-cellulose required to bind the specific binding proteins, and ligand specificity. The sedimentation properties of those 3-methylcholanthrene-binding proteins which were extracted with salt from DEAE-cellulose were examined on 5 to 20% sucrose gradients; the major binding species sedimented as a broad peak at 4.5 S. |
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Keywords: | 3-methylcholanthrene cytosolic binding protein assay DEAE-cellulose |
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