Kaufmann Stefan, Tanaka Motomu
Lehrstuhl für Biophysik E22 Technische Universität München James-Franck-Strasse 1, 85747 Garching, Germany.
Chemphyschem. 2003 Jul 14;4(7):699-704. doi: 10.1002/cphc.200200537.
This paper deals with single-step, orientation-selective immobilization of human erythrocyte membranes on bare silica beads with different topographies: 1) solid (nonporous) silica beads with a diameter of 3 microns and 2) porous silica beads with a diameter of 5 microns. Erythrocyte membranes were immobilized onto beads simply by incubation, without sonication or osmotic lysis. Membrane orientation before and after immobilization was identified with two immunofluorescence labels: 1) the extracellular part of glycophorin can be labeled with a first monoclonal antibody and a second polyclonal antibody with fluorescence dyes (outside label), while 2) the cytoplasmic domain of Band 3 can be recognized with a first monoclonal antibody and a second fluorescent polyclonal antibody (inside label). Adherent erythrocytes on the beads all ruptured, inverted the asymmetric orientation of the membrane, and selectively exposed their cytoplasmic domain. The surface topography did not influence the orientation or the amount of immobilized membrane. On the other hand, the fact that no adsorption or rupture of erythrocytes could be observed on planar quartz substrates suggests a significant influence of contact curvature on adhesion energy.
本文探讨了将人红细胞膜单步、定向选择性固定在具有不同形貌的裸露硅珠上的方法：1）直径为3微米的实心（无孔）硅珠和2）直径为5微米的多孔硅珠。红细胞膜只需通过孵育即可固定在珠子上，无需超声处理或渗透裂解。固定前后的膜取向通过两种免疫荧光标记来确定：1）血型糖蛋白的细胞外部分可用带有荧光染料的第一种单克隆抗体和第二种多克隆抗体进行标记（外部标记），而2）带3的细胞质结构域可用第一种单克隆抗体和第二种荧光多克隆抗体进行识别（内部标记）。珠子上附着的红细胞全部破裂，使膜的不对称取向反转，并选择性地暴露其细胞质结构域。表面形貌不影响固定膜的取向或数量。另一方面，在平面石英基板上未观察到红细胞的吸附或破裂这一事实表明接触曲率对粘附能有显著影响。
