Koch N, Haustein D
Mol Immunol. 1983 Jan;20(1):33-7. doi: 10.1016/0161-5890(83)90102-5.
Surface proteins of intact murine B lymphocytes were crosslinked by the bifunctional reagent, 4,4'-diphenyldiazoniumdisulphidfluoroborate and then radiolabelled with 125I. After solubilization of the cells, Ig-containing complexes (Ig and protein(s) covalently crosslinked to Ig) were isolated and analyzed by two-dimensional SDS polyacrylamide gel electrophoresis (2D-SDS-PAGE). Ig-containing complexes were separated in the first dimension by cylindrical SDS gels. Subsequently, the disulphide bridges of the isolated surface Ig molecules and of the crosslinking reagent were cleaved, and the products electrophoresed in the second dimension on SDS slab gels. In addition to the Ig polypeptide chains, two proteins with mol. wts of 46,000 and 56,000 could be identified. In order to answer the question whether these proteins are associated with IgM and/or IgD Ig-complexes were separated with regard to their isotype and analyzed separately by 2D-SDS-PAGE. It was found that both proteins are associated with subunits of IgM. In the case of IgD, no associated structures could be demonstrated by the method used.
完整小鼠B淋巴细胞的表面蛋白用双功能试剂4,4'-二苯基重氮二磺氟硼酸盐进行交联，然后用125I进行放射性标记。细胞溶解后，分离出含Ig的复合物（Ig和与Ig共价交联的蛋白质），并通过二维SDS聚丙烯酰胺凝胶电泳（2D-SDS-PAGE）进行分析。含Ig的复合物在第一维通过圆柱形SDS凝胶进行分离。随后，切割分离出的表面Ig分子和交联试剂的二硫键，并将产物在第二维的SDS平板凝胶上进行电泳。除了Ig多肽链外，还可鉴定出两种分子量分别为46,000和56,000的蛋白质。为了回答这些蛋白质是否与IgM和/或IgD相关的问题，根据其同种型分离含Ig的复合物，并通过2D-SDS-PAGE分别进行分析。发现这两种蛋白质都与IgM的亚基相关。对于IgD，所用方法未显示出相关结构。
