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標題: | 蛋白質二維電泳之鹽類效應與對策 Salt-induced artifacts in protein 2-dimensional gel electrophoresis and my strategy |
作者: | De-Yian Li 李德彥 |
出版年 : | 2002 |
學位: | 碩士 |
摘要: | “蛋白質體學”是大規模的檢視細胞、器官或生物體液中蛋白質的一門研究方法,而且需要許多嚴格控管的步驟。蛋白質體學的核心技術就是蛋白質二維電泳,到目前為止,還沒有其他技術有能力可以在一次的分離步驟中同時解析上千個蛋白質。 二維電泳由樣本處理開始,適當的樣本處理對於好的二維電泳是絕對必要的。理想的樣本處理結果應該要使蛋白質完全溶解、抑制結合、變性以及還原。而且蛋白質樣本中的非蛋白質的不純物可能幹擾電泳分離以及後續的二維電泳影像呈現,所以樣本處理應該要除去這些物質。 在鹽類的存在的情形下,第一維等電點聚焦電泳會有聚焦緩慢,可到達的最大電壓明顯的降低。此外,在高鹽濃度下進行等電點聚焦電泳所得到二維電泳的結果會有較差的解析度,以及部分由電泳造成artifacts的結果。而電泳產生的artifacts包含了有橫拖或者縱拖的斑紋,解析範圍內會有空白間隔,由於陰極漂移以及陽極漂移所造成pI解析範圍的喪失,更重要的是我們觀察到在高鹽濃度下進行等電點聚焦電泳會造成蛋白質不可逆的修飾反應。 當面臨鹽類效應所具有的高電導特性,我們提出一個可以解除這樣情況的方法。使用我們提出的膠體內透析的方法,可以大幅的解決上述的artifacts,此外還可以大量的增加等電點聚焦電泳的蛋白質負載量。在我們研究的過程中發現了一些以前所未定義的問題;陰極漂移和陽極漂移在鹽類效應的高電導度的情形下發生,而且與傳統的ampholyte帶出的pH梯度等電點聚焦電泳所發生的情形一致。另外;也有現象顯示有蛋白質修飾的傾向。然而等電點聚焦電泳所能到達的最高電壓下降的現像是可以排除的,因為總電導度是由最後的鹽濃度所決定,並且與其數值以及離子強度成正比。另外,污染的核酸會遮蔽蛋白質在低pH值範圍的解析。由於鹽類與核酸污染的蛋白質樣本會限制其最大的總蛋白質負載數量,膠體內透析可以提供一個移除鹽類與核酸的有效的方法,進而增加整個可以分析的蛋白質負載數量。 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75327 |
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顯示於系所單位: | 生化科學研究所 |
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