酵母因为有聚糖的壁而使其与细菌或动物细胞相比在某些方面变得比较棘手，比如重组质粒的转化，菌液PCR，蛋白提取，酵母质粒提取等，这些实验都需要考虑破壁问题，其中比较复杂的是蛋白提取。因为相对于酵母蛋白提取其它实验都有相对固定而又简便高效的方法，即使去网上随便一搜就能出来好多操作手册，自己根据实际情况加以改造也就很容易得到自己想要的结果。
但是对于蛋白提取来说，这方面的网上资料不是很多，中文文献所介绍的方法也各有不同，大都比较复杂。其中有一篇中文文献介绍并对比了几个国内外已发表的比较好的实验方法[1]。如果有感兴趣的同志可以看看。这篇文献采用的是蛋白双向电泳的方法比较提取蛋白效果，难怪对酵母细胞的处理不够简单。
我们如果仅仅做一下蛋白的Western blot，而且实验室没有那些罕见的的药品（比如酵母破壁的蜗牛酶，各种高大上的蛋白酶抑制剂等）怎么办？
其实对于酵母蛋白提取，我们主要关注两个问题（或两个点）就行了：1）酵母细胞的破壁；2）抑制很多内源性的蛋白酶。酵母细胞壁是很坚硬的，必须将物理的方法和化学的方法相结合。在提取蛋白上不推荐使用糖分解酶，因为它虽然能降解细胞壁上的糖聚合物，但也可能引入蛋白酶。在做实验时，只要关注上述那两个点，一般情况下Western blot都能得到好的效果。
下面介绍一下我的实验方法，该实验方法改自《酵母遗传学方法实验指南》，科学出版社，2000。
材料和溶液：
适量的生长培养基
50mmol/L Tris-HCl（pH7.5），10mmol/L 叠氮化钠
ESB（electrophoresis sample buffer）缓冲液（可在4℃贮存数天）
2% （w/v）SDS
80mmol/L Tris-HCl（pH6.8）
10% （v/v）甘油
1.5% （w/v）二硫苏糖醇（DTT）
0.1mg/ml （0.01%，w/v）溴酚蓝
配制方法（100ml），8ml的1M 的Tris-HCl，2g的SDS，10ml的甘油，1.5g的DTT，0.01g的溴酚蓝，用蒸馏水定容到100ml。在实际应用时，ESB用量很少，建议将各组分都先配成溶液后，再配ESB缓冲液。
操作步骤：
1) 用选择培养基（如SD/-Trp等）或一般培养基（YPD，YPDA）培养大量酵母细胞。培养基的选择对应于菌株的遗传状态和实验目的。一般液体培养的话，OD600要到1以上。
2) 取4mL 菌液高速离心，收集菌体，可以在1.5 ml或2 mL离心管中进行，多次离心富集，不离心时要置于冰上。建议高速快速离心（如13000rpm，15s左右）。这时可以将微波炉打开加热水了。
3) 加入200μL的50mM Tris-HCl，10mM NaN3溶液，重悬细胞，而后高速离心15s。
4) 弃上清，加入30μL 的ESB缓冲液，沸水煮5min，使蛋白酶失活之后，样品存放于-80℃5min。
5) 这时就可以用组织破碎仪暴力机械破碎了，有玻璃珠的加玻璃珠。我们实验室没有，我用的是3mm钢珠一个，每秒振荡30s，振荡2.5分钟。
6) 加入70μL ESB 缓冲液，稍加振荡，高速快速离心15s，沸水煮3min。
7) 高速离心15s，取10μL左右上清，上样进行SDS-PAGE凝胶电泳。因为我用的是大颗粒的钢珠（3mm，原操作方法采用的是0.2mm的玻璃珠）所以我离心后仍有大量沉淀，电泳时洗上清时一不小心就会吸到沉淀，但这些东西在电泳时会留在电泳加样口底部，对实验结果影响不太大，只要不太多就行了。
对于后续的Western blot，请见往期文章，对一个实验连续多做几次，边做边思考，不断改进和熟练，一定会有好的结果。
【提示】迅速煮沸样品可以防止蛋白质水解以保持其稳定。如果担心蛋白质被水解，可以在ESB中加PMSF从而减少蛋白质水解；对于极不稳定的蛋白质，有条件的实验室可以采用以下抑制剂：丝氨酸蛋白酶抑制剂，胃蛋白酶抑制剂A，E64等（具体根据实验而定）。
此处附上我的Western blot图，这两条带都是转有pGBKT7-53的Y2HGold酵母菌株表达后的条带。
另外给大家推荐两本资料
一个是上面提到的《酵母遗传学方法实验指南》（中文版），科学出版社，2000。
另一个是clontech公司的《Yeast Protocols Handbook》， clontech这本资料中给出了两种方法，但比较麻烦，如果你想要做一些更精确地实验推荐你看看。除了蛋白，这两本书对于你用酵母做其它实验也是很有帮助的，比如酵母双杂交和酵母文库构建等。这两本资料和文中的文献都可以在网上找到。
参考文献：
[1]（《酿酒酵母全蛋白的提取》，方芳，食品与发酵工业，2014）
作者，张昊伟，清华–伯克利深研院（TBSI）研究生（open fiesta项目）
图片来自网络，内容由作者原创，转载请注明！
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