多年的生化、遺傳學(xué)及分子分析研究所得的大量研究結(jié)果,使酵母成為生物制藥研究者首選的模式生物系統(tǒng)和工具。因此,酵母成為普遍的基因表達(dá)研究和重組蛋白的常見載體。盡管研究用的酵母種屬有多種,包括畢赤酵母屬、漢遜酵母屬和德巴利氏酵母屬,但最常用的還是酵母菌屬。
使用傳統(tǒng)的酶分解法通常很難從細(xì)胞中提取完整的酵母mRNA和細(xì)胞內(nèi)蛋白。裂解酶通常是粗制備,包含的RNA酶和蛋白酶不僅會破壞細(xì)胞壁,還會破壞目標(biāo)分子。而且,酶裂解出原生質(zhì)體通常需要加入去垢劑,而去垢劑會造成多種蛋白質(zhì)失活。因此,機械裂解或破壞酵母細(xì)胞通常需要釋放一些小分子。
1、破碎前的準(zhǔn)備:
0.1g菌體(本實驗以0.1g菌體為例);鋼珠;研磨機(選用品牌上海凈信科技Tissulyser-24);研磨罐。
2、研磨:
0.1g菌體加入30ML PBS溶液,混勻后用移液器均勻滴入液氮中,收集液氮凍住的小球,將小球放入上海凈信科技預(yù)冷過的研磨罐中,調(diào)整頻率,震蕩時間。取下研磨罐再次放入液氮預(yù)冷取出來再次上機研磨
研磨現(xiàn)場圖
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