Yang E B, Wang D F, Mack P, Cheng L Y
Department of Biochemistry, Faculty of Medicine, National University of Singapore, Crescent, Singapore.
J Biochem Biophys Methods. 1996 May 14;32(2):97-108. doi: 10.1016/0165-022x(96)00003-6.
In this work, a simple, sensitive, and non-isotopic assay system for the detection of EGF-induced EGF receptor degradation and tyrosine phosphorylation in intact cells is described. In this system, boiling Laemmli sample buffer was directly added to cultured Chang liver cells to stop the reactions in the cells stimulated by EGF and to make whole-cell extracts. The effects of EGF concentration and incubation time on the EGF-induced degradation and tyrosine phosphorylation of EGF receptor were successfully determined using monoclonal anti-EGF receptor, recombinant anti-phosphotyrosine peroxidase conjugate, and enhanced chemiluminescence (ECL) Western blotting assay system. Unlike other assay systems, the use of radioisotopes was avoided in this determination. The assay system is linear up to 100 micrograms sample protein from whole-cell extracts for the detection of EGF receptor and EGF-induced autophosphorylation. This assay may be easily adopted for identification of other growth factor receptors and phosphotyrosine-containing proteins in intact cells, using appropriate anti-growth factor receptor antibodies.
在本研究中，描述了一种用于检测完整细胞中表皮生长因子（EGF）诱导的表皮生长因子受体降解和酪氨酸磷酸化的简单、灵敏且非同位素检测系统。在该系统中，将煮沸的Laemmli样品缓冲液直接添加到培养的张氏肝细胞中，以终止EGF刺激的细胞中的反应并制备全细胞提取物。使用单克隆抗表皮生长因子受体、重组抗磷酸酪氨酸过氧化物酶偶联物和增强化学发光（ECL）蛋白质印迹检测系统，成功测定了EGF浓度和孵育时间对EGF诱导的表皮生长因子受体降解和酪氨酸磷酸化的影响。与其他检测系统不同，该测定避免了使用放射性同位素。该检测系统对于全细胞提取物中用于检测表皮生长因子受体和EGF诱导的自身磷酸化的样品蛋白，在高达100微克时呈线性。使用适当的抗生长因子受体抗体，该检测方法可轻松用于鉴定完整细胞中的其他生长因子受体和含磷酸酪氨酸的蛋白质。
