Casadei J M, Gordon R D, Lampson L A, Schotland D L, Barchi R L
Proc Natl Acad Sci U S A. 1984 Oct;81(19):6227-31. doi: 10.1073/pnas.81.19.6227.
A panel of 13 monoclonal antibodies against the voltage-sensitive Na+ channel of rat skeletal muscle has been characterized. Each of these antibodies reacted with the purified Na+ channel protein in a solid-phase radioimmunoassay. Nine antibodies specifically immunoprecipitated the Na+ channel in a form that retained its characteristic high affinity for saxitoxin, and 11 recognized the channel in a crude mixture of solubilized membrane proteins separated on a Sepharose CL-6B column. Six antibodies specifically labeled skeletal muscle in immunofluorescence techniques. In each case, antibody was localized only to the surface membrane of the muscle fibers. Eleven antibodies produced detectable reaction on immunoblot transfers of sarcolemmal membrane proteins; each of these bound to a diffuse 160- to 200-kDa band that comigrated with the large glycoprotein subunit of the purified Na+ channel. Further studies were carried out with one of these antibodies, L/D3. In immunoblots of a glycoprotein fraction prepared from muscle that had been homogenized rapidly in a solution containing detergent, EGTA, and protease inhibitors, L/D3 recognized only a single 260-kDa band. Incubation of solubilized muscle proteins at 4 degrees C for 24 hr without EGTA prior to isolation of the glycoprotein fraction resulted in partial conversion of this 260-kDa component to a smaller component between 160 and 200 kDa that comigrated with the principal immunoreactive component of sarcolemma. Based on its immunoreactivity with monoclonal antibodies, the large subunit of the rat skeletal muscle Na+ channel appears to be approximately equal to 260 kDa in its native state but may be sensitive to proteolysis during the isolation of sarcolemmal membranes.
已对一组针对大鼠骨骼肌电压敏感性钠通道的13种单克隆抗体进行了特性鉴定。在固相放射免疫测定中，这些抗体中的每一种都与纯化的钠通道蛋白发生反应。9种抗体以对石房蛤毒素保留其特征性高亲和力的形式特异性免疫沉淀钠通道，11种抗体在经琼脂糖凝胶CL - 6B柱分离的溶解膜蛋白粗混合物中识别该通道。6种抗体在免疫荧光技术中特异性标记骨骼肌。在每种情况下，抗体仅定位于肌纤维的表面膜。11种抗体在肌膜蛋白的免疫印迹转移上产生可检测的反应；这些抗体中的每一种都与一条弥散的160至200 kDa条带结合，该条带与纯化钠通道的大糖蛋白亚基一起迁移。用其中一种抗体L/D3进行了进一步研究。在由在含有去污剂、乙二醇双乙胺四乙酸（EGTA）和蛋白酶抑制剂的溶液中快速匀浆的肌肉制备的糖蛋白组分的免疫印迹中，L/D3仅识别一条单一的260 kDa条带。在分离糖蛋白组分之前，将溶解的肌肉蛋白在4℃下无EGTA孵育24小时，导致该260 kDa组分部分转化为160至200 kDa之间的较小组分，该组分与肌膜的主要免疫反应性组分一起迁移。基于其与单克隆抗体的免疫反应性，大鼠骨骼肌钠通道的大亚基在其天然状态下似乎约为260 kDa，但在肌膜分离过程中可能对蛋白水解敏感。
