发信人: nohate (无恨), 信区: Biology
标 题: Re: 蛋白纯化的脱盐问题
发信站: Unknown Space - 未名空间 (Sat Jun 19 23:30:34 2004) WWW-POST


The salt concentration after the gel filtration should be the same as the
running buffer. I usually use 100mM NaCl to run gel filtration, it depends on
the protein.
I don't know what you gonna do to store the protein you purified. I usually
concentrate the protein down to 10mg/ml, and save it for crystallization.
I save the protein in 5% Glycerol, 10mM DTT, 100mM NaCl and 20mM whatever
buffer. So I usually make the buffer, concentrate the protein down to a
reasonablly small volume, add the buffer, concentrate down again, add buffer
again, then concentrate down to 10mg/ml, then flash freeze in liquid nitrogen,
save in -80.
This process I call it buffer exchange, in your case, since you only need to
change the salt concentration, I would say this way is better, because you
will concentrate the protein anyway. The only thing you need to do is test the
best storage buffer for your protein.


【 在 capry (IA) 的大作中提到: 】
: Thank you for all the replies.
:
: My protein was first put in dialysis tubing, but somehow only a couples of
: hours later, most of them got precipitated. That is why I changed to
desalting
: column by which protein activity and stability are kept pretty good.
:
: The case is I will move on to do crystallization, so I would like to know by
: desalting column, what percent salt will be removed by estimation? You know
: salt is a very important consideration for protein crystallization.
:
: You can estimate the efficiency of dialysis tubing by simple calculation,
but
: I have no idea how to do that with desalting column. So I am asking for help
: here.
:
:
:
: 【 在 nohate (无恨) 的大作中提到: 】
: :
: : I usually do buffer exchange using concentrator, fast and easy.
: :
: : 【 在 forrestgump (阿甘) 的大作中提到: 】
: : : desalting column is much faster, but sometimes proteins bind to it
: : : non-specifically and you may lose some of your sample by that.
sometimes,
: : : proteins get denatured because the buffer change is too sudden, not like
: : : dialysis that is slow and "tender". plus, carry over is almost
inevitable
: on
: : : the column, and a 2nd run is sometimes necessary.
: : : dialysis on the other hand, has its disadvantage too. most of all, it
: takes
: : : way too long (O/N is routine), during which the protein may not be
happy.
: : : second, it dilutes ur sample to quite a bit (even more so than the
: column).
: : : u can read about this on those "current protocol of ..." books.
: : : anyway, if you have plenty of samples, i would go for the column.
: : :
: : : 【 在 comeandgo (春困秋乏夏打盹) 的大作中提到: 】
: : : : 用 5mM NH3HCO3 透析， 冻干时NH3HCO3也自动挥发
: : : : 【 在 capry (IA) 的大作中提到: 】
: : : : : Desalting column or dialysis bag, 哪个更彻底一些？有相应的文献资料吗
？
: : : : :
: : : : : 多谢
: : : : :
: : : : :
: : : :
: : :
: :
:
:

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