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[求助]
制备带有膜蛋白的膜组分用来做体外酶促反应
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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！ |
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3楼2013-09-29 14:33:18
克隆大虾
铁杆木虫 (著名写手)
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2楼2013-09-29 11:44:44
克隆大虾
铁杆木虫 (著名写手)
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【答案】应助回帖
感谢参与，应助指数 +1
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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
