蛋白质电泳_在蛋白质组学中的应用.ppt

表达上调 表达下调的点(right) 2DE常见问题及解决办法(Troubleshooting) Too much salt in the sample (disturbs IEF)Hint: Include an acetone precipitation to remove salts and other contaminants Charged impurities in the sampleHint: Include an acetone precipitation to remove salts and other contaminants Impurities in the sample or rehydration solutionHint: Include an acetone precipitation to remove salts and other contaminants Prepare new rehydration solution with pure reagents underfocused (Focusing time not long enough)Hint: Prolong the focusing time of the IEF Gel surface during polymerization not overlaid with destilled water (or too low amount of water used)Hint: Apply at least 1 ml to overlay the gel surface No uniform gel polymerization (e.g. impurities at gel cassettes, air bubbles in the polymerized gel)Hint: Clean gel cassettes with ethanol Cast the slab gel slowly (approx. 1 min) Overlay the gel carefully with destilled water Not all proteins (especially high molecular mass proteins) saturated with SDSHint: Use 0.15 % instead of 0.1 % (w/ v) SDS in the 1 x SDS buffer for SDSHigh sample load (protein disturbs separation of other spots or may not be fully saturated with SDS)Hint: Lower the protein amount Impurities on or within the 2-D gel still present during silver stainingHint: Clean the gel cassettes prior casting with ethanol Incubate the 2-D gels long enough (and with at least 100 ml/ gel) in fixing and washing solution prior staining 进行2DE分析几点建议 样品制备是关键，务必使蛋白样品充分裂解，除去颗粒性物质，如核酸、糖，盐及去污剂等的影响 要注意蛋白从第一向到第二向的转移，可用分离胶直接封闭IPG胶条 为了提高银染的效果，玻璃板和染色盘一定要清洗干净 如要进行差别蛋白组分析，银染的方法要注意和质谱分析相匹配 提高2DE分辨率的策略 首先采用宽pH范围的胶条，确定蛋白的分布范围，通常采用pH3-10的胶条 如果pH线性分布的胶条分离效果不好，可采用非线性胶条 加大蛋白上样量，提高局部蛋白的分辨率 采用窄pH范围的胶条，提高某pH范围蛋白的分辨率 第二向采用梯度胶进行分离 去除高丰度蛋白，进行蛋白的fractionation处理,富集低丰度蛋白 采用窄范围pH胶条对pH4-7的2D胶进行局部分辨率提高的分析举例 pH4-7胶条分析的2D胶图谱 pH4-5胶条分析的2D胶图谱 pH4-5胶条分析的2D胶图谱 pH5.5-6.7胶条分析的2D胶图谱 如上所示：通过分别用pH4-5,pH4-6,pH5.5-6.7的窄pH范围胶条，较单独用pH4-7的胶条，可大大提高局部区域的分辨率，使检测到的点数大大增加。 Pre-及subfractionation方法 Isolation of cel