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[求助]
制備帶有膜蛋白的膜組分用來(lái)做體外酶促反應(yīng)
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我現(xiàn)在要制備一個(gè)含有膜蛋白的memebrane fraction，用到的菌是E. coli(含帶有該蛋白基因的pET28質(zhì)粒)，誘導(dǎo)表達(dá)后收菌，超聲破細(xì)胞，5,000g離心10分鐘除去沒(méi)有破碎的細(xì)胞，然后再用70,000g離心收集膜組分，離心后在管底能看到透明的膜組分，用PBbuffer洗后再重懸在PB buffer中，用超聲打碎均一 請(qǐng)各位大俠指導(dǎo)，該方法有沒(méi)有問(wèn)題？因?yàn)殡x心后的膜是很粘的一整塊，所以要超聲很久才能打碎均一，能保證膜上蛋白的活性么？是否肉眼看起來(lái)打碎均一就行了，如果沒(méi)有打碎完全，膜是一小塊一小塊的，肯定會(huì)影響酶活，謝謝！ |
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克隆大蝦
鐵桿木蟲(chóng) (著名寫手)
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2樓2013-09-29 11:44:44
3樓2013-09-29 14:33:18
克隆大蝦
鐵桿木蟲(chóng) (著名寫手)
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【答案】應(yīng)助回帖
感謝參與，應(yīng)助指數(shù) +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樓2013-09-30 00:37:54
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