一个关于组织中膜受体蛋白提取的问题
丁香园论坛
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首先我的目的就是单纯的Western,要看的是一个膜受体的表达,以前我们做的方法是:提取组织(血管),加入lysis buffer,玻璃匀浆,冰上半小时,12,000rpm离心5min,取上清,加入loading buffer, 煮沸5min,上样........ 实验室所有的人做所有的蛋白都是这个步骤.从来也没有想过选择12,000rpm是什么道理. (大概认为提取的是总蛋白),在我做的过程中,感觉Western结果很不稳定,而且每次都显示这种受体含量很少似的,我想是否在12,000rpm后,我们所要的蛋白已经丢掉了,于是查了相关文献.发现了这样两种方法(与我们做的是同样的受体蛋白)
AT2 receptor–transfected or nontransfected COS-7 cells were extracted with lysis buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.02% sodium azide, 100 µg/mL PMSF, 1 µg/mL aprotinin, and 1% NP-40) and then centrifuged at 12 000g for 5 minutes. The supernatant was used for the analysis.
The rat kidneys, adrenal glands, and whole brains were dissected, minced, and homogenized in buffer A (10% glycerol, 20 mmol/L Tris-HCl, 100 mmol/L NaCl, 2 mmol/L PMSF, 2 mmol/L EDTA, 2 mmol/L EGTA, 10 mmol/L sodium orthovanadate, 10 µg/L leupeptin, and 10 µg/L aprotinin). The homogenate was centrifuged at 30 000g for 30 minutes at 4°C. The pellet was resuspended in buffer B (buffer A with 1% NP-40), stirred for 2 hours at 4°C, and centrifuged again at 30 000g for 30 minutes at 4°C. The supernatant was used for the analysis.
我们的方法和文献中用于细胞的方法相同.我不明白为什么细胞和组织蛋白提取的方法不同? 用文献中的方法提取的是总蛋白还是膜蛋白?有没有其他的提取蛋白的方法?希望高手帮助.
AT2 receptor–transfected or nontransfected COS-7 cells were extracted with lysis buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.02% sodium azide, 100 µg/mL PMSF, 1 µg/mL aprotinin, and 1% NP-40) and then centrifuged at 12 000g for 5 minutes. The supernatant was used for the analysis.
The rat kidneys, adrenal glands, and whole brains were dissected, minced, and homogenized in buffer A (10% glycerol, 20 mmol/L Tris-HCl, 100 mmol/L NaCl, 2 mmol/L PMSF, 2 mmol/L EDTA, 2 mmol/L EGTA, 10 mmol/L sodium orthovanadate, 10 µg/L leupeptin, and 10 µg/L aprotinin). The homogenate was centrifuged at 30 000g for 30 minutes at 4°C. The pellet was resuspended in buffer B (buffer A with 1% NP-40), stirred for 2 hours at 4°C, and centrifuged again at 30 000g for 30 minutes at 4°C. The supernatant was used for the analysis.
我们的方法和文献中用于细胞的方法相同.我不明白为什么细胞和组织蛋白提取的方法不同? 用文献中的方法提取的是总蛋白还是膜蛋白?有没有其他的提取蛋白的方法?希望高手帮助.
