公开(公告)号：US5741635A

公开(公告)日：1998-04-21

申请号：US593614

申请日：1996-01-30

申请人： Gerry R. Boss ,  Abhijit Guha ,  Jurgen S. Scheele ,  Anthony J. Pawson

发明人： Gerry R. Boss ,  Abhijit Guha ,  Jurgen S. Scheele ,  Anthony J. Pawson

IPC分类号： C07K14/47 ,  G01N33/574 ,  G01N33/68 ,  C07K1/00 ,  C12Q1/00

CPC分类号： C07K14/4722 ,  G01N33/57407 ,  G01N33/57415 ,  G01N33/57484 ,  G01N33/57496 ,  G01N33/6893

摘要： The invention features methods for measuring the total amounts of G protein-bound GDP and GTP in mammalian cells and tissue. The method of the invention is advantageous over conventional methods in that the cells need not be exposed to phosphate-free growth conditions, which can adversely affect the accuracy of the GTP determination. Moreover, the method does not require exposure of the cells to radiolabelled phosphate in the growth medium. Standardized amounts of G protein-bound GDP and GTP are determined relative to non-nuclear cellular protein content or cellular DNA content, allowing comparison of results across different cell types. The method is particularly applicable to the detection of Ras protein bound GTP and GDP. The determination of Ras-bound GTP levels by the method of the invention provides a sensitive and accurate means of evaluating the role of Ras activation in cellular processes, such as cell proliferative disease, hormonal secretion, cellular differentiation, programmed cell death, cell cycle regulation, and protein trafficking. Treatment of abnormal processes can then more accurately targeted to the alteration of G protein activation.

摘要翻译： 本发明的特征在于测量哺乳动物细胞和组织中G蛋白结合的GDP和GTP的总量的方法。 本发明的方法优于常规方法，因为细胞不需要暴露于无磷酸盐生长条件，这可能不利地影响GTP测定的准确性。 此外，该方法不需要将细胞暴露于生长培养基中放射性标记的磷酸盐。 相对于非核细胞蛋白质含量或细胞DNA含量确定G蛋白结合的GDP和GTP的标准化量，允许比较不同细胞类型的结果。 该方法特别适用于检测Ras蛋白结合GTP和GDP。 通过本发明的方法测定Ras结合的GTP水平提供了敏感和准确的方法来评估Ras激活在细胞过程中的作用，例如细胞增殖性疾病，激素分泌，细胞分化，程序性细胞死亡，细胞周期调节 ，和蛋白质贩运。 然后治疗异常过程可以更准确地针对G蛋白激活的改变。