发信人: moso (野鬼), 信区: Biology
标 题: Re: His-tag protein purification problem
发信站: BBS 未名空间站 (Wed Sep 13 01:26:29 2006), 转信

Well, before you try any of improvement, make sure that your processing
works
very well step by step, including:
1. the target protein is well expressed in your host system.
2. The his-taged protein is caught by your Ni-column or something else.
3. Every washing step doesn't wash the target off.

Only when you know where the problem is, you can reasonably make some
improvement. Like:
1. if you find that the protein expressed in a low efficiency, you should
get
the protein expression enhanced.
2. If the protein got difficulty in binding to the column, you may like to
conside to change the purification condition from nature to denatured
condition or reversely because of conformational concern. Well, only when
you don't have restriction in selecting purificaiton condition.
3. If you find you lost your protein during the washing, you need to use a
less
harsh condition to wash.
4. For the non-specific binding, 20mM or even higher starting Imidizole
could be of some help. It will reduce the non-specific binding. And
meanwhile you can
try a higher sodium chloride in your purification to cut down the non-
specific interaction.
5. I didn't see many that even 1M Imidizole fails to elute the his-taged
protein from the column. Usually it is because less protein was captured
by the column. Well, If it is really the case, you can consider to adjust
pH and salt concentration to a better condition for elution of your
target.



【 在 jinhua (jinhua) 的大作中提到: 】
: can someone please help me out? i am trying to purify a 150kD His tagged
: protien using Ni-NTA agarose beads. i am facing several problems-
: 1. a lot of non specific protein binding. tried washing with glycerol(10%)
,
: 20mM beta mercaptoethanol, and upto 80mM imidazole.
: 2. very low elution of the protein. even high concentration (1M) of
: imidazole does not help.
: Any help will be appreciated.



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