发信人: Marble (小石头哥哥), 信区: Biology
标 题: Re: 请教蛋白保存问题
发信站: The unknown SPACE (Sat Nov 16 17:08:59 2002), 站内信件

cystal of ice will form that can mechanically shear the protein.
the same as DNA. for storage of DNA, it is better to be stored
in buffer to avoid such mechanical shearing and hydrolysis,
if you concern about EDTA, you can use Tris-HCl (pH 8.0).
Here is some general guide for storage of purified proteins:
For short term storage (up to 24 h), most proteins can be kept
at 4C. For storage times longer than 24 h r at 4C, it may be
necessary to filter sterilize the protein preparation (through
a 0.22um filter) or to add a bacteriostatic agent (e.g. 0.1%
sodium azide) to avoid bacterial growth. Note that not all
proteins are stable at 4C for longer periods.
For long term storage (more than a week), it becomes necessary
to freeze the protein preparation. It is important to freeze
it rapidly using liquid nitrogen or a dry ice/ethanol mixture
to avoid denaturation. It is also important to freeze the
solution in small aliquots to avoid repeated freezing and
thawing which may reduce the biological activity or affect the
structure (e.g., antibodies, enzymes). Several stabilizing agents
can be added, such as glycerol (5-50% (w/v)), serum albumin
(10 mg/ml), reducing agents (such as 1 mM DTT), and ligands
(the nature and concentration depending on the nature and
concentration of the target protein).
If you want to store your protein preparation for several
months, you can store it at -20C. At this temperature it is
necessary to add 50% glycerol to the solution to avoid freezing.
The sample can be prepared in two ways:
Add an equal volume of pure glycerol to the protein preparation.
Or dialyze the protein preparation against the storage buffer
containing 50% glycerol. This method has the additional advantage
that it results in an approx. threefold concentration.
If you need to store your protein preparation for longer periods
(months to years), you should freeze it at -70C or even in
liquid nitrogen. Although it is not really necessary to add
glycerol at these temperatures, the addition of 5-50% glycerol
could help to keep the protein stable.
Alternative methods are:
Storage of the protein at 4C as an ammonium sulfate precipitate.
Storage of the protein at 4C or lower in a lyophilized form.
For the lyophilization it is necessary that the protein is
dissolved in a volatile buffer (such as trimethylamine/HCl;
pH range 6.8-8.8). Note that not all proteins are stable during
the freeze-drying process.
one more thing i should mention in supplement to this guide,
trace of protease will definitely degrade the protein, though
addition of protease inhibitors seems fine, it is suggested a
further purification is needed.


【 在 Rizhard (快乐的狗) 的大作中提到: 】
: why glycerol is good for protein?
: 【 在 mrp (mrp) 的大作中提到: 】
: : usually protein extract will be used in weeks, -80 is enough.
: : purified protein usually will be stored for a longer time, 10% glycerol
: : can stabilized the protein at -80.
: : if u want to store in -20, better add 50% glycerol, as most restriction
: : enzymes fomulation, also keeps protein in aqueous state.


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※ 修改:．Marble 于 Nov 16 17:14:43 修改本文．[FROM: 146.186.]
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 146.186.]