Detection of nonhuman primate gonadotropins in polyacrylamide gels: An alternative to the western blot

Authors

  • Dr. Robert L. Matteri,

    Corresponding author
    1. Wisconsin Regional Primate Research Center, University of Wisconsin, Madison
    • Wisconsin Regional Primate Research Center, University of Wisconsin, 1223 Capitol Court, Madison WI, 53715–1299
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  • Toni E. Ziegler

    1. Wisconsin Regional Primate Research Center, University of Wisconsin, Madison
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Abstract

The lack of appropriate antibodies restricts the use of western blots in studies of nonhuman primate gonadotropins. We now present the evaluation of an alternative method that can be applied in situations in which antibody is limiting or nonexistent. The use of nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (Swack et al., 1987) retains a significant degree of α-β subunit association, as determined by the western blot analysis of human and rhesus luteinizing hormone (LH; 33 and 36 kD, respectively). This observation was confirmed when eluates of gel slices were analyzed by specific gonadotropin immuno- and bioassays. The molecular weight of rhesus LH in pituitary protein was found to be 36 kD by bioassay and immunoassay. Similarly, the molecular weight of rhesus follicle-stimulating hormone (FSH) did not differ between assay systems (36 kD). The analysis of chorionic gonadotropin (CG)/LH activity in gel eluates of pregnant rhesus and tamarin urinary protein revealed major activity at 43 kD, with lesser bands between 34 and 39 kD. Guanidine-induced dissociation of gonadotropin subunits in pregnant rhesus urinary protein resulted in the disappearance of dimer at 43 and 36 kD and the appearance of 25 and 16 kD subunit peaks. This alternative to the western blot is not limited to SDS-PAGE. Native gel electrophoresis (no SDS) showed that rhesus FSH possesses a greater negative charge than rheus LH (Rf = 0.49 vs. 0.35). Isoelectric focusing PAGE resolved distinct isoforms of rhesus LH and FSH (pl range 5.2–7.2 and 4.0–5.8, respectively). The electrophoretic analysis of nonhuman primate gonadotropins in impure protein samples will provide fundamental data on the comparative biochemistry of these hormones. Applications will be found in biomedical and comparative studies.

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