Detection and microsequencing of juvenile hormone-binding proteins of an insect by the use of an iodinated juvenile hormone analog.
An [125I]iodinated juvenile hormone (JH) analog can be used as a sensitive and highly selective probe for the visualization of high-affinity, (JH)-specific binding proteins from insect hemolymph samples. The proteins can be detected in their native form using a two-dimensional (isoelectric focusing then native gradipore gel) separation of the crude protein mixture containing the 125I-labeled iodinated JH analog. The proteins can be transferred to activated glass fiber paper by electroblotting, and the location of the bound gamma-emitter can be found by exposure of the dried gel or the electroblot to X-ray film. The radiolabeled protein spot can be excised from the Coomassie-stained glass fiber paper and subjected directly to gas-phase N-terminal amino acid sequencing. This non-destructive, non-denaturing technique may have wide applicability in identifying and sequencing ligand-specific binding proteins in complex mixtures.
Kulcsár
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P
Department of Chemistry, State University of New York, Stony Brook 11794-3400.
Prestwich
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GD
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D006458:Hemolymph / Q000032:analysis; D019476:Insect Proteins; D007313:Insecta / Q000032:analysis; D007457:Iodine Radioisotopes; D007525:Isoelectric Focusing; D007605:Juvenile Hormones / Q000378:metabolism*
D002352:Carrier Proteins; D019476:Insect Proteins; D007457:Iodine Radioisotopes; D007605:Juvenile Hormones; C035606:juvenile hormone-binding protein, insect