Characterization of an antiserum to the rat luteal luteinizing hormone/chorionic gonadotropin receptor.
The LH/CG receptor was purified from detergent extracts of rat luteal tissue using hCG affinity and wheat germ agglutinin chromatography. Analysis of the purified material by silver staining of sodium dodecyl sulfate-polyacrylamide gels (with or without reducing agents) revealed a prominent broad band corresponding to a 93K protein and several minor contaminants. That the 93K band represents the LH/CG receptor is supported by the following. 1) This band is absent in material purified from rat luteal tissue in which the LH/CG receptor had been down-regulated. 2) [125I]iodohCG binding to Western blots of both the initial detergent extract and the purified material resulted in binding to a 93K protein. The 93K protein representing the LH/CG receptor was excised after sodium dodecyl sulfate-gel electrophoresis of the purified material and was electroeluted. Half of the electroeluted receptor was incubated with reducing agents. After mixing with nonreduced receptor, this mixture was used to immunize one rabbit. Immune, but not preimmune, serum (or immunoglobulin G purified thereof) recognized a 93K protein on Western blots of partially purified (approximately 10% pure) rat luteal receptor. No other bands were seen. Using this approach it was determined that the antiserum recognized reduced, nonreduced, denatured, and native forms of the receptor. A polyclonal antibody to the LH/CG receptor will be useful for studies on the structure and regulation of this receptor.
Rosemblit
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N
Population Council, New York, New York 10021.
Ascoli
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M
Segaloff
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DL
D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D006063:Chorionic Gonadotropin / Q000378:metabolism; D002845:Chromatography; D003338:Corpus Luteum / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007106:Immune Sera / Q000276:immunology*; D010084:Oxidation-Reduction; D051381:Rats; D011919:Rats, Inbred Strains; D011974:Receptors, LH / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism
D006063:Chorionic Gonadotropin; D007106:Immune Sera; D011974:Receptors, LH; C041222:iodo-chorionic gonadotropin