Hi Harvey,
Well, knowing nothing about your protein, allow me to ruminate anyway...
It sounds like you are exploring the possibility of a metal ion or
other cofactor being lost. This is a reasonable first thing to check,
but your buffer exchange steps should allow small cofactors (smaller
than most proteins that is) to pass through your membrane and away
from your protein. This suggests that your loss of activity is due to
the loss of something the size of your protein. Four things come to
mind right away.
1) The least exotic possibility I can think of is maybe your protein
is inactive all along according to your assay (your assay could have a
problem in it, I would suggest trouble shooting your assay as a first
step). This could result in your relatively dirtier prep falsely
reporting activity because of another protein component (i.e. an
impurity) that is active according to your assay, and then lost later
during your purification.
2) This next idea seems unlikely, but you asked so... Could there be
another protein component missing that is necessary for activity that
you don't know about? This protein would be lost during purification
resulting in an inactive form or your protein.
3) Probably another red herring here... Maybe your protein is not
stable without lots of other proteins around. I have personally seen
proteins that go to pot at low concentrations, but are very stable at
high concentrations, for which this sort of reasoning is invoked. You
could try adding Arg or other amino acids to keep it folded.
4) Is your protein active in a cleaved form? I have seen kinases with
competent kinase domains in the absence of regulatory domains. If you
run an activity assay that included the cleaved form of your protein,
and then lose this cleaved form later after purifying away the cleaved
protein, it would appear that you have lost activity.
The most important advice I can give you is to pay attention to what
your assays are really telling you, not what you think they are
telling you because of useful assumptions we all make, but what the
data really reports. For example, your activity assay shows no
activity, the problem could be your protein, or a component of the
assay, it is a bad idea to assume the protein is the only place
something could be wrong. A factual analysis will hopefully allow you
to trace back what you really know and where things could be going
wrong.
Hope this doesn't give you too many gooses to chase, hopefully
somewhere in here is a spark to help you reason yourself out of your
problem. Cheers~
~Justin
Quoting Harvey Rodriguez <[log in to unmask]>:
> Dear all,
>
> Recently, I came across an obstacle on the purification and acitivty
> measurement of my protein. My protein was expressed with an C terminal His
> tag in the HEK 293T cells and purified by nickel affinity, anion
> exchange and size exclucion chromatography. For every purification step, I
> preserved some sample to test the activty. Strikingly, the protein retains
> activity after nickel affinity column even for three days but lost almost
> all the activty immediately after Mono Q and SEC. Therefore, I speculated
> that something (metal ion or co-factor) binding to the protein was striped
> by the Mono Q column. Then I skipped this step and only use the SEC for
> further purification. However, the protein is still not active no matter
> what buffer I use, eg. Tris,Hepes or PBS. The protein I purified by nickel
> column is also in the PBS buffer and no additive was added. Buffer exchange
> in the concentrator doesn't affect the activity of the protein. Can anyone
> explain why anion exchange or size exclucion chromatography destroy the
> activity of the protein? Any comment or proposal is appreciated!
>
> Harvey
>
|