Establishment and optimization of two-dimensional electrophoresis for myocardial proteomes of mice
- VernacularTitle:小鼠心肌蛋白质组双向电泳技术的建立及其优化
- Author:
Ming LIU
;
Huijun WANG
;
Xuefeng LI
;
He JIN
;
Wei KANG
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2006;10(28):174-176,封三
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: Two-dimensional electrophoresis (2-DE) is precise and effective for the isolation and analysis of complex protein mixtures, has become one of valuable key techniques for proteome. Mice models were as research subjects to model human disease status, one of important methods for cardiovascular disease. The establishment and optimization of 2-DE for proteomes of mice myocardium will provide basis for heart disease.OBJECTIVE: To establish and optimize a stable technique of 2-DE for proteomic analysis of mice myocardium.DESIGN: Mice were used as research subjects, observational comparative study.SETTING: Department of Pathology, Nanfang Hospital Affiliated to Southern Medical University.PARTICIPANTS: The experiment was performed at the Department of Pathology, Nanfang Hospital Affiliated to Southern Medical University between February and September 2004. Totally 12 male BABL/c mice of 4-5 weeks without special-pathogen free with the body mass of 12-15 g were selected, which was provided by the Experimental Animal Center of Southern Medical University.METHODS: The mice were put to death by dislocation of cervical vertebra under drugged state to obtain heart. Various protein extraction method,loading sampling buffer, strip range of IPG, sampling volume of protein,staining method and so on were compared, analyzed, scanned and digilized,and then performing image analysis. Each link in the study was established and optimized.MAIN OUTCOME MEASURES: Resolution of protein and reproducibility of the trial.RESULTS: A method that was optimal for cardiac tissue can isolate about a total of 910 protein spots on pH 4-7 gel strips. 200 μg was fit for silver stain while 1000 μg was for Coomassie stains. It was found that in the myocardium of normal mice about (920±30) protein spots were obtained in 17 em pH 3-10 L gel strip, while around (880±30) were gained in the pH 4-7 L gel strip. The mean matching rate achieves to 86. 9%.CONCLUSION: The findings of the trial showed that 2-DE technique can be effectively applied for proteomics of myocardium of mice to regulate and optimize, and relatively stable separation method of myocardial protein was established.
