发信人: royluo ( 罗马情结), 信区: Biology
标 题: Re: a question for protein expression in pichia
发信站: The unknown SPACE (Sat Dec 28 19:38:08 2002), 站内信件

你最好做个阴性对照看看那些条带是不是杂蛋白。
我觉得75KD的那条带很有可能只是杂蛋白吧。之所以
你能在WB上看到这条带应该是因为它量太大，二抗
与它有非特异性结合的原因。
糖基化不可能让24KD的SIZE增大那么多，况且
你用的pichia 比较低等，只能加一些最简单的N-LINK
糖基在上面。
BTW：我们实验室也有人做这个系统。也是表达
HUMAN或者MOUSE的蛋白。你们有没有表达什么酶过呢？
如果有活性如何呢？


【 在 nkbabie (Nankai) 的大作中提到: 】
: I expressed one mouse gene in Pichia by secreting vector pPICZa. But when I
: run SDS-PAGE using the supernatant, there were many bands, from ~22 kDa to
: ~75kDa. my expected protein size is ~24 kDa. The 75 kDa band was strongest. In
: Western blot, I could see strong band at the expected position (24 kDa), but
: also could see relative weak band around at 75 kDa(I don't whether it is
: specific or nonspecific).
: I am wondering why it showed so many bands. My expectation is only one. Even
: if it is glycosylated, I think 70 kDa is too big.
: Thanks for your help.


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