From: rashmi panigrahi
Date: 1 March 2012 13:05
Hi all,
1)
I have a protein which gives two peaks on the 1ml Histrap column, Has anyone seen this kind of behaviour 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
----------
From: Artem Evdokimov
Hi,
Sounds like your proten gradually aggrgates. Seems tat aggregation is exacerbated by disulphide crosslinking. There might also be a conformational change involved or perhaps one of your dimers is a 'true' dimer (for a given value of true) whilst the other one might be a disulphide- or metal- mediated. I would not mix the imac peaks, no. Some thoughts: attempt different resins for imac which allows you to play with imidazole concentrations for elution (some resins elute as low as 80-100 mM). Add edta right after elution. Add tcep instead of bme and see if things change. Have you already attempted gentle proteolysis? Do you have an activity assay?
Good luck,
Artem
----------
From: Pius Padayatti
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
----------
From: Vandna Kukshal
hi rashmi ,,,
--
Vandana kukshal
Date: 1 March 2012 13:05
Hi all,
1)
I have a protein which gives two peaks on the 1ml Histrap column, Has anyone seen this kind of behaviour 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
----------
From: Artem Evdokimov
Hi,
Sounds like your proten gradually aggrgates. Seems tat aggregation is exacerbated by disulphide crosslinking. There might also be a conformational change involved or perhaps one of your dimers is a 'true' dimer (for a given value of true) whilst the other one might be a disulphide- or metal- mediated. I would not mix the imac peaks, no. Some thoughts: attempt different resins for imac which allows you to play with imidazole concentrations for elution (some resins elute as low as 80-100 mM). Add edta right after elution. Add tcep instead of bme and see if things change. Have you already attempted gentle proteolysis? Do you have an activity assay?
Good luck,
Artem
----------
From: Pius Padayatti
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??
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.
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
----------
From: Vandna Kukshal
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.
Vandana kukshal
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