Protein Extraction from Solid Tissue
Protein Extraction from Solid Tissue
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DOI：
10.1007/978-1-59745-423-0_17
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发表时间：
2011-01-01
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影响因子：
--
通讯作者：
Nister, Monica
中科院分区：
文献类型：
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作者：
Ericsson, Christer;Nister, Monica
从患病或健康组织中最大限度地提取和溶解蛋白质对于使整个蛋白质补体可用于蛋白质组学分析是重要的。它还有助于最大限度地提高可重复性并最大限度地减少浪费。蛋白质氨基酸骨架的最小降解或去磷酸化对于保持提取物的分析实用性至关重要。样品的密封对于最大限度地降低样品污染的风险非常重要。通过冷冻崩解/基于SDS的溶解方法，所提出的蛋白质提取和溶解的标准方案可导致脑组织98%的溶解，相当于约100 μ g蛋白质/mg组织湿重：通过用无菌钢球摇动，在冷冻管中将组织在冷冻状态下压碎。粉碎后，在70 ℃下，以相当于组织湿重10倍的体积，在2%SDS中振荡提取和溶解10分钟。低温管中的密封有助于防止污染。用SDS样品缓冲液处理可以抑制蛋白酶和磷酸酶活性。所得蛋白质提取物可用于SDS PAGE、2-D PAGE、Western印迹、ESI-MS和Rasa。建议的标准协议有可能找到广泛的应用，蛋白质提取，溶解，鉴定和定量从冷冻保存的临床样品是可取的。
Maximal extraction and solubilization of protein from diseased or healthy tissue is important to make the whole protein complement available for proteomic analysis. It also helps to maximize reproducibility and to minimize waste. Minimal degradation of the protein amino acid backbone or dephosphorylation is essential to preserve the analytical utility of the extract. Containment of the sample is important to minimize the risk of contamination to and from the sample. The proposed standard protocol for protein extraction and solubilization can result in 98% solubilization of brain tissue, corresponding to about 100 mu g protein per mg tissue wet weight, by a frozen disintegration/SDS-based solubilization method: Tissue is crushed in the frozen state in a cryotube by shaking with a sterile steel ball. The crushing is followed by the extraction and solubilization in 2% SDS for 10 min, at 70 degrees C, in a volume corresponding to ten times the tissue wet weight, with shaking. The containment in a cryotube helps to prevent contamination. The treatment with SDS sample buffer can inhibit protease and phosphatase activity. The resulting protein extracts can be used for SDS PAGE, 2-D PAGE, Western blotting, ESI-MS, and RASA. The proposed standard protocol has the potential to find wide application where protein extraction, solubilization, identification, and quantitation from cryopreserved clinical samples are desirable.