Salt-induced artifacts in 2-dimensional gel electrophoresis and my strategy

• Main Authors: Li De-Yian, 李德彥
• Other Authors: Chang Geen-Dong
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/58737389253301130967

碩士 === 國立臺灣大學 === 生化科學研究所 === 90 === Abstract “Proteomics” is the large-scale screening of the proteins of a cell, organism or biological fluid, a process which requires many stringently controlled steps. The core technology of proteomics is 2-dimensional (2-D) electrophoresis. At present, there is no other technique that is capable of simultaneously resolving thousands of proteins in one separation procedure. The 2-D process begins with sample preparation. Proper sample preparation is absolutely essential for a good 2-D result. Ideally, the preparation process will result in the complete solubilization, disaggregation, denaturation, and reduction of the proteins in the sample. Non-protein impurities in the protein sample can interfere separation and subsequent visualization of 2-D result, therefore sample preparation should rid the sample of these substances. First dimension focusing in the presence of salt is slower and the maximum attainable voltage is considerably lower. In addition, poor resolution of 2-D electrophoresis and artifacts of electrophoresis result from high salt concentration. The artifacts include streaking in both longitudinal and horizontal directions, gaps, the absence of focusing, cathodic drift and anodic drift. More importantly, we observed that irreversible protein modification reactions occurred in the presence of salts in isoelectric focusing (IEF). To alleviate the artifacts associated with high salts in IEF, we introduced a transient in-gel dialysis procedure. Using this proposed procedure, the artifacts mentioned above were largely removed. In addition, the sample loading capacity for IEF can be increased by the in-gel dialysis procedure. Some undefined questions came up during the study of this thesis. The cathodic drift and the anodic drift occurred under high salt concentration, which is in agreement with classic carrier ampholyte pH gradient gel electrophoresis. In addition, we observed a tendency of protein modification when IEF was conducted with samples of high salt concentrations. Contaminated nucleic acids would screen protein resolution near the low pH region. The contaminated salts and nucleic acids of the protein sample limit the maximal loading capacity. In-gel dialysis can eliminate the contaminated salts and nucleic acids and enhance the total loading capacity of sample.