圓點墨點分析法作為篩選窖蛋白一在胃AGS細胞異位表現之快速且有效的工具

• Main Authors: Chia-Wei Tsu, 許家瑋
• Other Authors: Hua-Wen Fu
• Format: Others
• Language: en_US
• Online Access: http://ndltd.ncl.edu.tw/handle/10324219338598798230

碩士 === 國立清華大學 === 分子與細胞生物研究所 === 96 === Caveolin-1 is a scaffolding protein that mediates caveolae formation and gives rise to cellular responses by interacting with caveolae-localized signal molecules. The various level of caveolin-1 expressed in gastric cancer (GC) cells is associated with oncogenesis. Besides, the oncogene-induced cell transformation reduces the tyrosine phosphorylation status of caveolin-1. To further investigate the association of expression of caveolin-1 and its phosphorylation status with oncogenesis, caveolin-1 and phosphorylation-deficient caveolin-1/Y14F mutant were introduced into gastric cancer AGS cells. Here, the dot blot analysis was chosen to achieve time-saving, large-scale, generic-to-use, and low-complexity in screening and quantification of the expression of caveolin-1 and caveolin-1/Y14F in the collection of AGS transfectants. After analysis by dot blot, 9 out of 11 and 7 out of 11 of AGS transfectants stably express caveolin-1 and caveolin-1/Y14F, respectively. Moreover, with respect to the quantification of caveolin-1 expression in AGS transfectants, the dot blot analysis showed results similar to those yielded by the western blot analysis. However, the dot blot analysis showed about 5-fold higher than western blot analysis in quantification of the amount of caveolin-1/Y14F. The differences in the quantification of caveolin-1/Y14F between the dot blot and western blot analyses were due to the decrease in binding affinity of anti-caveolin-1 antibody to the caveolin-1/Y14F. Although the amount of caveolin-1/Y14F expressed in AGS transfectants is needed for further investigation, dot blot analysis can serve as an efficient tool for screening and quantification of the amount of caveolin-1 expressed in AGS transfectants. Dot blot should also be able to be applied in screening the expression or knockdown of caveolin-1 in other cell lines.