A radioimmunoblotting method for measuring myosin light chain phosphorylation levels in smooth muscle.
A radioimmunoblotting method for measuring myosin light chain phosphorylation levels in smooth muscle.
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用于测量平滑肌中肌球蛋白轻链磷酸化水平的放射免疫印迹方法。
DOI：
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发表时间：
1985
影响因子：
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通讯作者：
J. Haeberle
中科院分区：
文献类型：
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作者：
D. Hathaway;J. Haeberle
建立了一种测量完整平滑肌肌球蛋白轻链磷酸化的摩尔化学计量学方法。牛主动脉平滑肌的20，000-Da轻链的抗血清从兔中收获，并用于通过放射免疫印迹程序标记轻链。在最初的表征中，发现20，000-Da轻链可以通过电印迹从聚丙烯酰胺凝胶转移到硝酸纤维素纸上，在0.1-5.0微克的蛋白质范围内效率约为80%。在1：500的稀释度下，未纯化的轻链抗血清在22 ℃下需要约10-12小时以达到与转移的轻链的平衡结合。此外，用125 I-蛋白A平衡标记轻链-抗体复合物需要在22 ℃孵育4-6 h。通过使用这些条件，开发了20，000-Da轻链的放射免疫测定，其在0.1-5.0微克（5-250 pmol）的蛋白质范围内呈线性。如果使用第二抗体程序（山羊抗兔免疫球蛋白G Fab片段），则可以测量低至20 ng的轻链。磷酸化和非磷酸化的肌球蛋白轻链用甘油-尿素聚丙烯酰胺凝胶电泳分离。这个程序，结合放射免疫印迹，得到类似的估计磷酸化水平相比，直接测定磷酸盐或扫描考马斯亮蓝染色的凝胶。此外，当应用于完整的子宫平滑肌，甘油-尿素凝胶放射免疫印迹得到的值的肌球蛋白轻链磷酸化的放松和收缩的肌肉，没有统计学差异，从那些获得的二维电泳方法。（250字处删节）
A method for measuring the molar stoichiometry of myosin light chain phosphorylation in intact smooth muscle has been developed. Antiserum to the 20,000-Da light chains of bovine aortic smooth muscle was harvested from rabbits and used to label light chains by a radioimmunoblotting procedure. In the initial characterization it was found that the 20,000-Da light chains could be transferred by electroblotting from polyacrylamide gels to nitrocellulose paper with an efficiency of approximately 80% over a protein range of 0.1-5.0 micrograms. At a dilution of 1:500, the unpurified light chain antiserum required approximately 10-12 h at 22 degrees C to reach equilibrium binding to the transferred light chains. Moreover, equilibrium labeling of the light chain-antibody complex with 125I-protein A required 4-6 h of incubation at 22 degrees C. By using these conditions, a radioimmunoassay for the 20,000-Da light chains was developed that was linear over a protein range of 0.1-5.0 micrograms (5-250 pmol). As little as 20 ng of light chains could be measured if a second antibody procedure (goat anti-rabbit immunoglobulin G Fab fragments) was used. Phosphorylated and unphosphorylated myosin light chains were separated by glycerol-urea polyacrylamide gel electrophoresis. This procedure, combined with radioimmunoblot, gave similar estimates of phosphorylation levels when compared with direct assay for phosphate or scanning of Coomassie blue-stained gels. Moreover, when applied to intact uterine smooth muscle, the glycerol-urea gel radioimmunoblot gave values of myosin light chain phosphorylation for relaxed and contracted muscles that were not statistically different from those obtained with a two-dimensional electrophoretic method.(ABSTRACT TRUNCATED AT 250 WORDS)