急!酵母中半乳糖诱导表达
最近用BY4741转化pESC-Leu载体做表达, 在SD缺Leu的培养基上筛选,2天后能够看到大量很明显的转化子(用加水的BY4741感受态做对照不长)。但是用半乳糖诱导,破菌后SDS-PAGE跑上清沉淀,看不到目的蛋白,空载体和质粒跑得SDS-PAGE差不多结果。初步推测应该是没有诱导成功。尝试过下列几种诱导方式:
1.SD培养基上转化子转到YPD培养基摇过夜, 转移到大量YPD培养基再次摇过夜,离心得到菌体再转移到YPG培养基上,16度诱导过夜。其中OD600监测不低于0.6.
2.转化子转到液体SD培养基摇过夜,离心,菌体中加入SG培养基诱导过夜
3.转化子 接到SD(无galactose), YP(无galactose)两种培养基中分别培养1小时后,加棉子糖和galactose做诱导培养
诱导温度都是16度过夜,也试过一次25度6小时
基本上都看不到目的蛋白~
求助大神 到底是哪里除了问题?或者是不是需要做western验证?
请各位不吝赐教,感激不尽~~~ 返回小木虫查看更多
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同问,我这个是pESC-HIS载体,PCR验证质粒转进去了,就是30摄氏度,140rpm, D-半乳糖诱导不生长,不知道哪有问题
补充可以看看这个https://www.thermofisher.com/cn/ ... etting-started.html
Some researchers choose to grow yeast in medium containing 2% galactose as the sole carbon source during induction. However, yeast typically grow more quickly in induction medium containing 2% galactose plus 2% raffinose. Raffinose is a good carbon source for yeast, and unlike glucose, does not repress transcription from the GAL promoter. Raffinose is a trisaccharide of galactose, glucose, and fructose linked in that order. Most yeast can cleave the glucose-fructose bond, but not the galactose-glucose bond. Fructose is then used as a carbon source.也有见过加甘油和半乳糖,
请问,你最后诱导出来了吗?
我跟你做的一样,半乳糖诱导可以生长,但是一直没有诱导出目的蛋白。
您好,请问您最终做出来了吗?我想向您请教一下。