| 查看: 786 | 回复: 3 | ||
[求助]
制备带有膜蛋白的膜组分用来做体外酶促反应
|
|
我现在要制备一个含有膜蛋白的memebrane fraction，用到的菌是E. coli(含带有该蛋白基因的pET28质粒)，诱导表达后收菌，超声破细胞，5,000g离心10分钟除去没有破碎的细胞，然后再用70,000g离心收集膜组分，离心后在管底能看到透明的膜组分，用PBbuffer洗后再重悬在PB buffer中，用超声打碎均一 请各位大侠指导，该方法有没有问题？因为离心后的膜是很粘的一整块，所以要超声很久才能打碎均一，能保证膜上蛋白的活性么？是否肉眼看起来打碎均一就行了，如果没有打碎完全，膜是一小块一小块的，肯定会影响酶活，谢谢！ |
回复此楼» 猜你喜欢
OpenAI又攻克数学难题了，离AI做有机合成还有多远？
已经有12人回复
-
极限拉扯，如何抉择
已经有13人回复
-
课题组会帮你通过考核么？
已经有7人回复
-
入职后第一次管课题组的专利缴费，对账才发现官费比别人多掏了一倍
已经有6人回复
-
售一区SCI文章T0P，我:8O.551.O54,科目全,可十急
已经有9人回复
-
售SCI一区T0P文章，我:8.O55.1.O.54,科目全,可十急
已经有8人回复
-
售SCI一区文章，我:8O5.5.1.O5.4,科目全,可伽急
已经有10人回复
-
售SCI一区T0P文章，我:8.O55.1.O.54,科目全,可十急
已经有9人回复
-
售SCI一区文章，我:8.O.55.1.O.54,科目齐全,可伽急
已经有7人回复
-
售SCI文章，我:8O5.5.1.O.54,科目齐全,可+急
已经有10人回复
克隆大虾
铁杆木虫 (著名写手)
- 应助: 133 (高中生)
- 金币: 6581.9
- 散金: 236
- 红花: 10
- 帖子: 2377
- 在线: 1348.2小时
- 虫号: 93868
- 注册: 2005-09-15
- 性别: GG
- 专业: 微生物生理与生物化学
2楼2013-09-29 11:44:44
3楼2013-09-29 14:33:18
克隆大虾
铁杆木虫 (著名写手)
- 应助: 133 (高中生)
- 金币: 6581.9
- 散金: 236
- 红花: 10
- 帖子: 2377
- 在线: 1348.2小时
- 虫号: 93868
- 注册: 2005-09-15
- 性别: GG
- 专业: 微生物生理与生物化学
【答案】应助回帖
感谢参与，应助指数 +1
|
Expression and purification of the membrane proteins All three membrane proteins were cloned from genomic DNA into standard expression vectors. Expression was performed in prokaryotic cells using standard protocols. The cells were harvested at 3000g and resuspended in lysis buffer containing protease inhibitor tablets (Roche). After lysis using a cell disruptor (Constant Cell Disruption Systems), unbroken cells were removed by centrifugation at 8000g for 15 min. Membranes were harvested from the resulting supernatant by ultracentrifugation for 1 h at 100,000g. For each of the target proteins, the membranes were solubilized in 1% DDM. Insoluble material was then removed by ultracentrifugation at 100,000g for 1 h. All steps following cell harvest were carried out at 4°C; all purification buffers contained 300 mM NaCl. The solubilized proteins were individually purified by one-step Ni2+-NTA (Qiagen) affinity chromatography. The fractions containing the purified target proteins, as assessed by SDS-PAGE, were pooled prior to UDS. The concentration of the protein was confirmed using the BCA assay (Pierce). |
4楼2013-09-30 00:37:54
