发信人: ouba (comfortably numb), 信区: Biology
标 题: Re: 请教NMR 样品准备问题
发信站: BBS 未名空间站 (Mon Aug 8 22:16:20 2005)



【 在 crystalmaker (风雨无阻) 的大作中提到: 】
: 晶体长不出来，决定试试NMR。
: 有几个问题：
: 1。样品量：0.5mM， 500 ul，够吗？
: 分子量13K,6.5mg/ml,0.5 ml,3.5-4 mg,够吗?
should be fine. for regular nmr tubes, 400ul is okay.
if u buy a shigemi tube, 250ul is enough. higher concentration
will help if ur protein doesn't complain.


: 2.标记问题:C13和N15
: 大家从哪儿买这样的LB medium?还是单买自己混合?
: 请推荐质量好的厂家.
we buy from cambridge isotope. u can't use LB medium but need
miminal medium instead.Some people use bioexpress but i don't
think it's worth the price unless ur protein doesn't express
in miminal.

: 3.表达量:
: 在这种特殊的medium中,细菌的表达量会不会降低?我原来是一升可以出1mg,不知道
: 换了C13和N15的medium表达量会不会象Se-Met那样变低.
Maybe, have to try to know.
The whole process will usually take several months depending on the protein.
It helps to assign the backbone and do a heteronuclear noe on N-H which
tells you the flexible part on ur protein. if it's on the termini, u may
try truncating it and try crystalizing the shorter protein.

: ...................



--

※ 来源:·BBS 未名空间站 http://mitbbs.com·[FROM: 68.235.]