hi rashmi ,,,
I have one suggestion dont use KCL in your buffer for
crystallization b'coz u ll get lots of salt crystals mostly of K2SO4 . i
faced this problem for one of my halophilic protein . most of the condition
where NH4So4 is there u ll get salt crystals.
On Thu, Mar 1, 2012 at 8:48 PM, Pius Padayatti <ppadaya...@gmail.com> wrote:
> 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
> <rashmi.panigrah...@gmail.com> 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
>
--
Vandana kukshal
