Rashmi,
>Has anyone
> seen this kind of behaviour?
Yes, have seen differentially Glycosylated protein samples elute off
affinity resins as separate peaks.
but not that they behave different on gelfiltration like you described
in your case.
possible the samples interact with resin
> Will there be problem if I mix the two peaks and load on the S-75 column??
yes , i will not mix the two peaks, especially if the aim is to crystallize.
or at least until i know what is going on.
>I added bME (2mM) after the protein came out of superdex, and there was no
> precipitation.
Keep DTT or TCEP (better) throughout your preparation if that helps.
but try TCEP instead and add it more often depending on its half life.
add a different salt. is there any reason you use KCl instead of NaCl?
Use a phosphate based buffer instead.
Add detergent to your buffer (triton or NP-40 or any detergent that
will not affect your protein activity, to a certain extent your
activity will be affected, but 20% reduction is ok)
Try Idoacetamide treatment
before binding to the resin. 2mM final and incubate your crude sample
for minimum of
2hrs.( especially if your protein is rich in disulfides)
Try a different type of homogenization method.
The other details you wrote here seems very confusing and not worth discussing.
Since your sample in the begining was not good as the days went by
the results you saw was probably not very worth paying attention to.
you will never even able to see those behavior in a future prep.
I would strongly suggest do very small pilot preps that can be
finished quickly in a day or two.
and try all the different things that are suggested here and by others.
and make sure you do try things same always but one parameter at a time.
It is very desirable to have an assay (binding, activity,
thermostability etc) throughout your
experiment.
hope this helps
On Thu, Mar 1, 2012 at 8:05 AM, rashmi panigrahi
<[log in to unmask]> wrote:
> Hi all,
> 1)
> I have a protein which gives two peaks on the 1ml Histrap column,r and does this mean that there are two
> populations of protein. They are partially seperated.
> 2)
> I tried to load the two peaks seperately on the superdex-75pg column.
> They came out as roughly dimer but the difference in the peaks is 6mls
> According to calculation with gel filtration standards
> one was 1.8mer
> and the other was 2.3 mer
> Will there be problem if I mix the two peaks and load on the S-75 column??
> 3)
> The protein is in 50mM HepespH7.3, 500mMKCl and 10% glycerol and imidazole
> when it comes from the NiNTA column,
> It is loaded on the superdex with same buffer but no imidazole. I get the
> dimer peak.
> If I concentrate and leave it, it start precipitating the next day even at
> 2mg/ml.
> I added bME (2mM) after the protein came out of superdex, and there was no
> precipitation.
>
> Hence for the next prep, I did the superdex run with bME in the buffer,
> there was a dimer peak and a small peak coming at the 125mls which is more
> than 1CV (S-75 is a 120 ml column). Loaded this small peak on the gel and it
> gave the same size band my protein. It could be my protein ...
>
> Hence I took the dimer from the above run concentrated and reloaded back on
> the same column and there was a very tiny peak for dimer which was 10 mAU
> for 0.5mls which is very less comapred to what I loaded.
>
> Does it mean that my protein is unfolded because of bME???
>
> Any idea to stop precipitation would be helpful.
>
> with regards
> Rashmi
>
>
>
--
Pius S Padayatti,PhD,
Phone: 216-658-4528
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