A modified quantitative EMSA and its application in the study of RNA--protein interactions.
Electrophoretic mobility shift assay (EMSA) is used to detect the complex of protein and nonradioisotope-labeled probe qualitatively. In this paper, we describe a modified quantitative EMSA, which uses biotin-labeled RNA in the complex formation. The RNA-protein complex is separated by agarose gel electrophoresis and capillary transferred to a positively charged nylon membrane. It is then detected through a secondary detection system using 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-nitroblue tetrazolium (NBT) as the colorimetric precipitating substrate. After scanning and quantification by an image analysis program, ImageQuant, it was observed that the optical density of the bands was proportional to the decadic logarithm value of standard RNA quantities in the tested range. By using the standard curve of the optical densities plotted against the logarithm values of RNA quantities in the linear range, we can calculate RNA quantities according to the optical density of the band and make a quantitative analysis of EMSA. This method was applied successfully in the study of the interactions between the AU-rich element (ARE) and HuR, which is one of the human members of the (embryonic lethal abnormal vision) ELAV family. The results reveal that the biotin-labeled AUFL transcripts in the RNA-protein complex can be detected quantitatively, while maintaining the biological features of its binding ability to the HuR protein. By this method, it is possible to conduct qualitative and quantitative analyses of the EMSA in the study of RNA-protein interactions.
Li
Yue
Y
Health Science Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai Research Center of Biotechnology, 500 Caobao Road, Shanghai 200233, PR China.
Jiang
Zhaozhao
Z
Chen
Haixu
H
Ma
Wei-Jun
WJ
D000469:Alkaline Phosphatase / Q000378:metabolism; D000954:Antigens, Surface / Q000737:chemistry; D001703:Biophysics / Q000379:methods*; D001710:Biotin / Q000737:chemistry; D019411:Clinical Laboratory Techniques; D004305:Dose-Response Relationship, Drug; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D019075:Electrophoresis, Capillary; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007211:Indoles / Q000494:pharmacology; D009580:Nitroblue Tetrazolium / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000032:analysis*; D012313:RNA / Q000032:analysis* / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry
D000954:Antigens, Surface; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; C098770:ELAVL1 protein, human; D007211:Indoles; D011506:Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D009580:Nitroblue Tetrazolium; C035455:5-bromo-4-chloro-3-indoxyl phosphate; D012313:RNA; D001710:Biotin; D005982:Glutathione Transferase; D000469:Alkaline Phosphatase