Stanker L H, McKeown C, Balhorn R, Lee C, Mazrimas J, Goralka M, Wyrobek A
Lawrence Livermore National Laboratory, University of California, Livermore.
Mol Reprod Dev. 1992 Dec;33(4):481-8. doi: 10.1002/mrd.1080330415.
High molecular weight proteins in Rattus norvegicus that are immunoreactive with an anti-protamine 2 specific antibody but not with an anti-protamine 1 specific antibody are described. These proteins were detected by coupling high-performance liquid chromatography (HPLC) with an enzyme-linked immunosorbent assay (ELISA). Briefly, following HPLC separation of rat sperm nuclear proteins, the HPLC fractions were probed with the antibodies. We estimate that the antibody probes are 100-1000 times more sensitive than UV absorbance measurements. Immunoblot analysis following acid-urea electrophoretic separation of rat sperm nuclear proteins, and of the HPLC fractions, also detected putative protamine 2 precursor proteins. The proteins reactive with the anti-protamine 2 antibody are most likely not mature protamine 2, since they were detected in a region of the chromatogram where we would not expect protamine 2 to migrate based on the chromatographic locations of human and mouse protamine 2. Likewise, the immunoblotting experiments demonstrated that the anti-protamine 2 antibody recognized proteins with slower electrophoretic mobilities than would be expected for a mature protamine 2. An anti-protamine 1 monoclonal antibody, Hup1N, that binds rat protamine 1 is also described. Hup1N allowed for identification of the HPLC fractions that contained rat protamine 1. Finally, we demonstrated that Hup1N binds protamine 1 from a large number of species, suggesting a conserved epitope for Hup1N.
描述了褐家鼠中与抗鱼精蛋白2特异性抗体发生免疫反应但不与抗鱼精蛋白1特异性抗体发生免疫反应的高分子量蛋白质。这些蛋白质通过高效液相色谱（HPLC）与酶联免疫吸附测定（ELISA）联用进行检测。简而言之，在对大鼠精子核蛋白进行HPLC分离后，用抗体检测HPLC馏分。我们估计抗体探针的灵敏度比紫外吸收测量高100 - 1000倍。对大鼠精子核蛋白以及HPLC馏分进行酸脲电泳分离后的免疫印迹分析，也检测到了假定的鱼精蛋白2前体蛋白。与抗鱼精蛋白2抗体发生反应的蛋白质很可能不是成熟的鱼精蛋白2，因为它们是在色谱图的一个区域中检测到的，基于人和小鼠鱼精蛋白2的色谱位置，我们预计鱼精蛋白2不会迁移到该区域。同样，免疫印迹实验表明，抗鱼精蛋白2抗体识别的蛋白质电泳迁移率比成熟鱼精蛋白2预期的要慢。还描述了一种结合大鼠鱼精蛋白1的抗鱼精蛋白1单克隆抗体Hup1N。Hup1N可用于鉴定含有大鼠鱼精蛋白1的HPLC馏分。最后，我们证明Hup1N能结合多种物种的鱼精蛋白1，这表明Hup1N存在一个保守表位。
