Weak binding of His-tags to IMAC

Hi all,

I am having difficulty getting much of my His-tagged protein complex to bind
IMAC beads (I'm using Talon which has immobilised Co2+). Most of my protein
comes out in the washes at pH 8.0 and only a small portion comes out when I
elute with either imidazole or EDTA. My troubleshooting guide suggests a
higher pH might help but 8 is already quite high. I am also using 300 mM
NaCl in my wash buffers, could this be the problem? Any ideas appreciated.

Cheers,
--
Bean

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Historians believe that in newspost <4330f46c$[Only registered users see links. ].au> on
Wed, 21 Sep 2005, Bean Long <[Only registered users see links. ].edu.au> penned the
following literary masterpiece:

If the HIS tag is buried in your protein and therefore not accessible,
then nothing you do with buffers will have any affect - it will not
bind.

Duncan
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they go flying by.

Duncan Clark
GeneSys Ltd.

"Bean Long" <[Only registered users see links. ].edu.au> wrote in
news:4330f46c$[Only registered users see links. ].au:


My his-tagged protein, too, binds relatively weakly to resins. You just
have to try different resins and conditions. I now use histrap_HP columns
from what is now GE Healthcare, in part because we got the chance to
purchase an HPLC machine ... I do not use any imidazole in the binding or
washing, but still have to really check peak purity (still in the process
of getting problems solved).

--
Best regards
Han
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Depending on how desperate you are, you might start trying different
metals on your column (e.g., Cu2+ or Zn2+). I think Cu is supposed to
give stronger binding (sometimes too strong?).
--
__________________________________________________ ____________________________
Lou Hom >K'93
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Historians believe that in newspost <dgrv67$28kv$[Only registered users see links. ]>
on Wed, 21 Sep 2005, Louis Hom <[Only registered users see links. ]> penned the
following literary masterpiece:

Using IDA-sepharose (Amersham's Chelating -sepharose) we quite often
found that Co2+ is better than Ni2+, in terms of binding capacity and
specificity of elution.

Duncan
--
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.

Thanks for all the replies. I can get weak binding both under native and
denatured conditions, so I don't believe that accessibility is a problem. I
have several other IMAC resins to try and will have a go with them today.

Many thanks,

--
Bean

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"Duncan Clark" <[Only registered users see links. ].com> wrote in message
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In article <4330f46c$[Only registered users see links. ].au>, "Bean Long" <[Only registered users see links. ].edu.au> wrote:

My observation:

The bigger is the protein and the more acidic is the protein, the less
its His-tagged form binds to Ni-NTA. I had one case where 7His would
quantitatively elute at 20 mM imidazole...

I don't think it has anything to do with the tag being buried/bound
and not exposed enough - in all of these cases I had
no trouble at all cleaving the tag off. Also, I purified one very acidic
protein (no tag!) by binding it to another one 6His-tagged protein.
The tagged partner alone elutes at ~ 150 mM imidazole - that is,
tag is fully exposed and works fine. But the complex elutes at 30 mM
imidazole. Crystal structure of the complex is solved and I don't
think there is a room there for the tag suddenly becoming buried.

One would think it's the case of some sort of ionic repulsion
(I *think* Ni-NTA should have residual negative charge (?)) but
salt does not appear to diminish this effect - at 600 mM the
difference in binding between "working" and "non-working" cases
is as serious as at 250 mM.

The size dependence is not as easy to define - it's just been
a consistent trend I observed over the course of many years and
many proteins. I explain it mechanistically (need stronger force
to hold bigger mass) but I continue to be puzzled by the acidity
effect.

Dima

Thanks Dima, This makes sense. My tagged protein is part of an extremely
large complex which is possibly too big to be held stationary on the beads
without some "leakage" I'm woring on ways to overcome this.

Cheers,
--
Bean

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"DK" <[Only registered users see links. ]> wrote in message
news:dgucm4$5f7$[Only registered users see links. ].wisc.edu...
<[Only registered users see links. ].edu.au> wrote:
bind
protein
I
appreciated.

In article <4333395e$[Only registered users see links. ].au>, "Bean Long" <[Only registered users see links. ].edu.au> wrote:

I do have couple suggestions to try:

1. Switch to Pi or hepes, pH ~ 7.0- 7.2 rather than tris 8.0. It sounds
cunterintuitive but with 150 K very acidic protein that won't bind
tightly, the binding was clearly less at pH 8.0 than at 7.0.

2. You are using highest specificity combination (Talon-Co2+).
In this case, highest specificity = lowest affinity. Try Ni-NTA
or, perhaps much better solution, Ni-IDA. You may have more
contaminants but that OK - poor purification is better than no
purification at all.

3. Forget His-tag, clone in Strep-tag and purify on Streptactin.
It is of much higher affinity and specificity interaction, so much
better purity is almost guaranteed. Expsnsive but in the case of
large complexes might be worth it.

Dima

Hi Dima,

I just tried Bio-Rad's Profinity today and the binding was, relative to the
result on Talon, fantastic. Same buffers except for addition of a little
glycerol. I'll keep working on this to see if I can optimise. We have a
couple of other tags but not a Strep-tag. Will discuss it with the boss.
Many thanks for your feedback.
--
Bean

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"DK" <[Only registered users see links. ]> wrote in message
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<[Only registered users see links. ].edu.au> wrote:
extremely
beads