Weber M M, Spöttl G, Gössl C, Engelhardt D
Medical Department II, Laboratory of Endocrine Research, Klinikum Grosshadern, University of Munich, Germany.
J Clin Endocrinol Metab. 1999 May;84(5):1679-84. doi: 10.1210/jcem.84.5.5686.
The insulin-like growth factor (IGF)-binding proteins (IGFBPs) from adult human serum, amniotic fluid, and cerebrospinal fluid were analyzed by a modified two-dimensional gel electrophoresis followed by Western ligand blotting. The samples were subjected to immobilized pH gradient isoelectric focusing in the first dimension, followed by nondenaturing SDS-PAGE in the second dimension and autoradiography after ligand blotting with [125I]IGF-I or [125I]IGF-II. The identity of the binding proteins was confirmed by immunoblotting and immunoprecipitation with specific antibodies. Using this method, all six human high affinity IGFBPs could be clearly separated from each other according to their molecular mass and isoelectric points (pI). All IGFBPs exhibited a variety of specific pI isoforms, which presumably represent posttranslational modifications. In adult human serum, glycosylated IGFBP-3 is found as a broad band of spots with molecular masses of 41 and 45 kDa and a pI in the range of 4.8-8.2. The two IGFBP-3 bands could be reduced to a single 36-kDa band after deglycosylation (pI 6-9). Furthermore, the specific spots for IGFBP-2 (33 kDa; pI 6.2-7.1) and deglycosylated IGFBP-4 (24 kDa; pI 6.3, 6.5, and 6.8) were found with their expected molecular masses. Additionally, the diffuse bands around 30 kDa, found in one-dimensional Western ligand blotting, could be clearly separated into distinct groups of specific spots representing IGFBP-1 (30 kDa; pI 4.0-4.8), IGFBP-6 (30 kDa; pI 4.8-5.8), glycosylated IGFBP-4 (29 kDa; pI 6.1 and 6.3), and IGFBP-5 (30/31 kDa; pI 6.4-8). As expected, IGFBP-6 was visible only when IGF-II was used as radioligand. In conclusion, two-dimensional gel electrophoresis followed by Western ligand blotting allows identification of all six high affinity IGFBPs with their isoforms on the basis of their characteristic molecular masses and pI, especially in the range of 30 kDa. This technique can be rapidly performed with small amounts of complex biological fluids and is a powerful tool for the detection and analysis of posttranslational modifications of IGFBPs.
采用改良的二维凝胶电泳结合 Western 配体印迹法，对成人血清、羊水和脑脊液中的胰岛素样生长因子（IGF）结合蛋白（IGFBP）进行分析。样品首先在第一维进行固定化 pH 梯度等电聚焦，然后在第二维进行非变性 SDS-PAGE，并用[125I]IGF-I 或[125I]IGF-II 进行配体印迹后进行放射自显影。通过用特异性抗体进行免疫印迹和免疫沉淀来确认结合蛋白的身份。使用该方法，可根据其分子量和等电点（pI）将所有六种人类高亲和力 IGFBP 彼此清晰分离。所有 IGFBP 均表现出多种特定的 pI 同工型，推测这代表翻译后修饰。在成人血清中，糖基化的 IGFBP-3 表现为一条宽带状斑点，分子量为 41 和 45 kDa，pI 在 4.8 - 8.2 范围内。去糖基化后（pI 6 - 9），两条 IGFBP-3 带可还原为单一的 36 kDa 带。此外，还发现了具有预期分子量的 IGFBP-2（33 kDa；pI 6.2 - 7.1）和去糖基化的 IGFBP-4（24 kDa；pI 6.3、6.5 和 6.8）的特异性斑点。另外，在一维 Western 配体印迹中发现的 30 kDa 左右的弥散带，可清晰分离为代表 IGFBP-1（30 kDa；pI 4.0 - 4.8）、IGFBP-6（30 kDa；pI 4.8 - 5.8）、糖基化的 IGFBP-4（29 kDa；pI 6.1 和 6.3）以及 IGFBP-5（30/31 kDa；pI 6.4 - 8）的不同特异性斑点组。正如预期的那样，仅当使用 IGF-II 作为放射性配体时才能看到 IGFBP-6。总之，二维凝胶电泳结合 Western 配体印迹能够基于其特征分子量和 pI 鉴定所有六种高亲和力 IGFBP 及其同工型，特别是在 30 kDa 范围内。该技术可使用少量复杂生物流体快速进行，是检测和分析 IGFBP 翻译后修饰的有力工具。
