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Detection and microsequencing of juvenile hormone‐binding proteins of an insect by the use of an iodinated juvenile hormone analog
Author(s) -
Kulcsár Peter,
Prestwich Glenn D.
Publication year - 1988
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(88)80582-9
Subject(s) - electroblotting , juvenile hormone , hemolymph , isoelectric focusing , chromatography , biochemistry , coomassie brilliant blue , chemistry , amino acid , polyacrylamide gel electrophoresis , biology , hormone , enzyme , staining , genetics
An [ 125 I] 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 125 I‐labeled iodinated JH analog. The proteins can be transferred to activated glass fiber paper by electroblotting, and the location of the bound γ‐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.