发信人: visa (有卡人士), 信区: Biology
标 题: Re: 谁有经验？ 关于dimerization 和 native gel
发信站: Unknown Space - 未名空间 (Tue Apr 13 14:43:44 2004) WWW-POST

【 在 nohate (无恨) 的大作中提到: 】
:
: I used to make different constructs for 1 protein, they are all soluble, but
: behave different, some are monomer, inactive, some are dimer inactive, some
: are dimer active can't crystallize, some are dimer active can
: crystallize......
: And the difference between these constructs are just around 10 residues.
~~~~~~~~~~~
this difference is already big enough, for my protein, a Ser-->Ala will kill
it all.
I am not sure I know you situation well, but I would suggest you to focus on
one
construct at a time or plan your constructs with less different residues. I am
working on enzyme mechanism, so I am very allergic to point mutations, hehe.
10 residues
different at active site with no significant harm to activity, for me, that is
a miracle.

:

: 【 在 visa (有卡人士) 的大作中提到: 】
: : I would like to suggest that whether it is dimer or not fully depends
: : on condition, I have one protein mutant that when purified by nickle
: : column,a2b2 complex is got, but run in native gel, dissociation is
observed
: : it is because of ionic strength difference between nickle column buffer
: : and my native gel running buffer. as I know, when they are making crystal
: : very extrem condition is used, so I would suggest you try a different
: : condition which close to crystallization to see if there is dimer
: :
: : 【 在 littlecrab (小赖子乖乖) 的大作中提到: 】
: : : E coli. 表达纯化的yeast蛋白不能形成二聚体 （GST pulldown), 但是crystal
: : : stucture 显示有 dimer 的可能。那么也许某种yeast in vivo
的修饰是形成dimer
: 所不
: : : 可缺少的, E. coli 则没有这种修饰。
: : : 所以我致力于验证in vivo dimer. Co-IP worked. 但是是over-expression. 我需
要
: 另一
: : : 个approach 佐证。那位大哥有这方面经验呢。如果直接load　whole cell
extracts
: : : 于non-denaturing gel, 然后做western, 可行吗？
: : : 如果先做IP, 有没有办法再跑native gel 呢？好像没法把目的蛋白从beads 上洗
脱
: 而不
: : : 破坏dimer 啊。
: : : 还有别的什么建议吗。注意，in yeast, 即使是overexpression,
: : : 也不可能看到coomassiee 染色的带的.
: : : 千万帮帮我啊。我遇到别人提问，我答得上的都答的－－－不许到前面翻，我会的
本
: 来就
: : : 不多。
: :
: :
:

--
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