Abstract:

BACKGROUND: Two-dimensional electrophoresis (2-DE) separation technology is one of core technologies for proteomics studies. However, the resolution of 2-DE map is highly altered due to different experimental conditions. Therefore, a 2-DE map with high resolution can be obtained via the optimization of key experimental conditions for a definite proteome sample.
OBJECTIVE: To establish an effective separation system of 2-DE for the proteome of HK-2 human renal tubular epithelial cells.
METHODS: HK-2 human renal tubular epithelial cells were cultured routinely. The proteins were extracted from HK-2 cells after lysing, and separated by 2-DE on the standard experimental condition with an optimization for each important experimental parameter. Electrophoresis parameters recommended by Bio-Rad Corporation were adjusted adequately. In the process of silver staining, a sufficient washing operation ensured a weak background of 2-DE maps. The gray densities and numbers of protein spots, resolution and contaminating strands in 2-DE maps were determined.
RESULTS AND CONCLUSION: A 2-DE separation system for proteome of human renal tubular epithelial cells was successfully established. The optimized experimental conditions were selected finally as follows: The lysis buffer containing 1% TBP, 4% CHAPS, 0.2% Bio-Lyte, 40 mmol/L Tris, 8 mol/L urea, 2 mol/L thiourea was applied to extract proteins from cells, and the passive rehydration proposal of samples was used to help the separation of large molecular weight proteins on the pH 4-7 IPG gels. Electrophoresis parameters recommended by Bio-Rad Corporation were adjusted adequately. In the process of silver staining, a sufficient washing operation ensured a weak background of 2-DE maps. Under the optimized conditions, 2-DE maps with high resolution have been reproducibly obtained.