熟悉提蛋白的大侠帮助一下
丁香园论坛
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要做肌浆网上的一蛋白,用RIPA试剂来从组织里提取;按它的说明如下:
1.每100mg组织加入1mlRIPA和10ulPMSF(不能预先加到RIPA种,否则PMSF失效)。样本以玻璃匀浆器匀浆,埋在冰里,间或匀浆,避免起泡沫和发热,冰里匀浆;
2.将样本转移到1.5ml离心管,4度高速离心30分钟。高速20000g低温离心。
3.将上清小心转移到干净无菌离心管中,-20度保存。
4.抽提的样本蛋白浓度可以用Lowry 法。空白对照采用RIPA。
上周看到一文献用如下方法,系同一蛋白。
1.Freshly dissected and minced tissues, were homogenized with an Ultra-Turrax T25 homogenizer in ten volumes of homogenization buffer (10 mM Hepes, pH 7.2, containing 0.25 M sucrose, 0.5 mM EDTA, 1 mM dithiothreitol, 0.2 mM PMSF, 1 mM benzamidine, 10 µM leupeptin, 1 µM pepstatin A and 100 nM aprotinin).
2.Homogenates were centrifuged for 25 min at 4000 g (Beckman JA20) and the supernatant re-centrifuged for a further 60 min at 100000 g (Beckman 50.2Ti).
3. Microsomal pellets were resuspended in four volumes of homogenization buffer by using ten strokes of a Dounce homogenizer, then rapidly frozen and stored at -80 °C until required. All procedures were performed at 4 °C.
文献的方法更好的原因是它提的蛋白范围更窄吗?
buffer不一样,会对提的蛋白有影响吗?哪个配方更好,我买的RIPA标称是最适合提动物蛋白的,和它的比不知如何?
如果我想换用文献的差速离心,而不换buffer可以吗?
就是用RIPA+PMSF来匀浆,最后一步溶解pellets也用RIPA吗?不是的话,那用什么溶解合适呢?
还有,我问过用100000g的超速离心,他们有专用的10ml管子,但我的上清很少,才1ml多,用什么介质溶液加满才行呀。
1.每100mg组织加入1mlRIPA和10ulPMSF(不能预先加到RIPA种,否则PMSF失效)。样本以玻璃匀浆器匀浆,埋在冰里,间或匀浆,避免起泡沫和发热,冰里匀浆;
2.将样本转移到1.5ml离心管,4度高速离心30分钟。高速20000g低温离心。
3.将上清小心转移到干净无菌离心管中,-20度保存。
4.抽提的样本蛋白浓度可以用Lowry 法。空白对照采用RIPA。
上周看到一文献用如下方法,系同一蛋白。
1.Freshly dissected and minced tissues, were homogenized with an Ultra-Turrax T25 homogenizer in ten volumes of homogenization buffer (10 mM Hepes, pH 7.2, containing 0.25 M sucrose, 0.5 mM EDTA, 1 mM dithiothreitol, 0.2 mM PMSF, 1 mM benzamidine, 10 µM leupeptin, 1 µM pepstatin A and 100 nM aprotinin).
2.Homogenates were centrifuged for 25 min at 4000 g (Beckman JA20) and the supernatant re-centrifuged for a further 60 min at 100000 g (Beckman 50.2Ti).
3. Microsomal pellets were resuspended in four volumes of homogenization buffer by using ten strokes of a Dounce homogenizer, then rapidly frozen and stored at -80 °C until required. All procedures were performed at 4 °C.
文献的方法更好的原因是它提的蛋白范围更窄吗?
buffer不一样,会对提的蛋白有影响吗?哪个配方更好,我买的RIPA标称是最适合提动物蛋白的,和它的比不知如何?
如果我想换用文献的差速离心,而不换buffer可以吗?
就是用RIPA+PMSF来匀浆,最后一步溶解pellets也用RIPA吗?不是的话,那用什么溶解合适呢?
还有,我问过用100000g的超速离心,他们有专用的10ml管子,但我的上清很少,才1ml多,用什么介质溶液加满才行呀。