摘要
目的对用于磷酸化蛋白质的相对定量分析的一种基于双向差示凝胶电泳(2-DDIGE)技术和Pro-Q Diamond荧光染色的方法进行考察。方法采用DIGE技术,从Pro-Q Diamond染色后对Cy2、Cy5荧光强度的影响、Pro-Q Diamond染色过程中采用的试剂对CyDye标记的蛋白质的影响、去离子水对DIGE扫描结果的影响、CyDye染料扫描的稳定性影响等方面,考察了该方法的有效性和特异性。结果在Pro-Q Diamond染色前对DIGE凝胶进行处理的过程中,有多种因素影响荧光强度变化,以致在进行Pro-Q Diamond染色后,凝胶点的荧光强度变化无法区分是染色造成的灰度值变化还是CyDye染料自身的变化,从而使通过荧光强度变化来定量检测磷酸化蛋白质变得困难。结论不宜采用对同一DIGE凝胶进行Pro-Q Diamond再染色检测磷酸化蛋白质的方法进行比较蛋白质组学的定量分析,为能同时展示出胶上的磷酸化蛋白质和总蛋白质,可通过DIGE实验与Pro-Q Diamond分开运行,再通过DIGE扫描图谱与Pro-Q Diamond染色图谱进行匹配的方法进行改进。
Objective To investigate a phosphoproteome relative quantitative analysis method based on 2-D DIGE and fluorescence staining. Methods By the DIGE technique, the effect of Pro-Q Diamond on fluorescence intensity of Cy2 and Cy5, the effect of some reagents during the staining, the effect of deionized water on the scanning, and the scanning stability of CyDye staining were all investigated. Results Before the Pro-Q Diamond staining, the intensity fluorescence was changed during the procedure of DIGE could be affected by many factors. So the reason of the intensity changes could be due to the staining or the CyDye itself, therefore increased the difficulty of the subsequent quantitative analysis of phosphoproteins. Conclusion A relative quantitative analysis method based on 2-D DIGE following fluorescence staining was not suitable for phosphoprotein analysis. To analysis phosphoproteins and the total proteins at the same gel, a separated procedure of DIGE and Pro-Q Diamond should be performed followed by a match between a scanning map and a Pro-Q Diamond staining map.
出处
《热带医学杂志》
CAS
2012年第12期1434-1437,F0004,共5页
Journal of Tropical Medicine
基金
国家自然科学基金(81102410)
