发信人: littlecrab (小赖子乖乖), 信区: Biology
标 题: Re: GST-Fusion 蛋白提纯求助!!!
发信站: Unknown Space - 未名空间 (Mon Mar 8 22:15:36 2004) WWW-POST

He meaned you don't have to add glycerol at all if you find it does not hurt
the protein property. If you know it is an enzyme, I think you had better add
glycerol, because freezing and thawing cycle can kill the enzyme. For other
proteins, I do not bother to add glycerol either.

And to get rid of gluathione, besides dialysis, there is an easy way. Take a
spin-column with an appropriate cut-off filter, e.g. 30KD, spin, concentrate
the protein and get rid of the solvent in the meanwhile. Then add the solvent
you want, spin again. Repeat if you want. Millipore company sell these columns
with different cut-off.


【 在 horizon (东方之子) 的大作中提到: 】
: Thanks! in your opinion, for the storage, you prefer adding equal amount of
: 50% glycerol not equal amount of pure glycerol?
: 【 在 Genesis (LIH) 的大作中提到: 】
: : if the protein is purified as GDT-fusion, you must have glutathione in the

: : prep, then depending on your subsequant application, you may want to
dialyze
:
: : it using whatever buffer you used to purify it minus glutathione.
: :
: : the storage question is trickier. if the purified protein is stabel, you
can
:
: : certainly keep it in -80, but some of them are finicky. temperature shift
: may
: : cause aggregation and precipitation after freeze. my feeling is, in
protein
: : manupilation, try to keep as little additives such as glycerol in the prep
: : as poosible, if you can get away without adding glycerol, I'd just freeze
it
:
: : withhout.
: :
: : the binding assay, if it uses the GST as binding modual, you certainly
want
: : to get rid of the glutathione. if it's used to bind to other protein, then
: : it really depends, you have to test the condition to see if glutathione
: : interferes with the binding.
: :
: : so the key is, do as little as you can to preserve the protein. that's
just
: : my way of dealing with protein.
: :
: :
: : 【 在 horizon (东方之子) 的大作中提到: 】
: : : 我正在表达，提纯一个GST-Fusion 蛋白，有几个问题想求教大家一下。
: : : 我是用一个Kit来purify的，但不知purify后要不要dialysis一下再保存?? 我想保
存
: 长一
: : : 些时间，-20度还是-80度好? 加等体积甘油还是50%甘油?
: : : 能不能不加甘油就直接把GST-Fusion蛋白冻存在-20度和-80度???
: : : 我能不能直接用kit purify出来的GST-Fusion 蛋白来做binding
: : : assay，还是要dialysis一下才行?
: : : 关于dialysis，以前我做过His-tag的dialysis，但不知是不是一样?
: : : 哪里能找到dialysis的配方或做dialysis的protocol??
: : : 不胜感谢!!!
: :
: : r
: :
:
:


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