Protein precipitation by storage - (Oct/13/2013 )
Hi,
I have stored my recombinant protein at 4°C for 2 weeks after dialysis. The protein is precipitated. But when vortexing it is dissolved again! have I lost my protein? Is it degraded?Please help me with this problem.
It really depends on your prior experience with the same protein in the same buffer.
In general, you could check for degradation by running a gel and looking out for strange bands that haven't been there before (degradation products). If the degradation is severe, you will either see no bands at all (degradated into very small fragments that elute from the gel) or a smear.
Of course it could be that upon precipitation your protein was denaturated. In that case, it _might_ theoretically re-dissolve (though it might as well not, really depends on your protein) and give you a band of the correct size. But your protein's activity might not be there any more. If you have a readout assay for the activity of your protein, use that to verify sample quality.
If the activity is there and the protein band(s) look normal, everything should be alright.
If you don't have an activity assay or if you are in any doubt, I would rather discard the protein and get a new aliquot rather than second-guessing your downstream applications.
Good luck,
map3k
map3k on Mon Oct 14 09:35:56 2013 said:
It really depends on your prior experience with the same protein in the same buffer.
In general, you could check for degradation by running a gel and looking out for strange bands that haven't been there before (degradation products). If the degradation is severe, you will either see no bands at all (degradated into very small fragments that elute from the gel) or a smear.
Of course it could be that upon precipitation your protein was denaturated. In that case, it _might_ theoretically re-dissolve (though it might as well not, really depends on your protein) and give you a band of the correct size. But your protein's activity might not be there any more. If you have a readout assay for the activity of your protein, use that to verify sample quality.
If the activity is there and the protein band(s) look normal, everything should be alright.
If you don't have an activity assay or if you are in any doubt, I would rather discard the protein and get a new aliquot rather than second-guessing your downstream applications.
Good luck,
map3k
Hi,
Thanks for your reply.This problem just happened to one of the dialyzed batch and the other one is OK.. Today I run my protein on the gel and I saw the band of the expected size. It seems that it is OK. My protein is the fusion of a ribosomal and a cell surface proteins. Since my protein is not an enzyme, should i check it for the activity?
Mariam on Mon Oct 14 17:39:23 2013 said:
Since my protein is not an enzyme, should i check it for the activity?
Well I don't know about your downstream application. If your protein of interest does not have enzymatic activity, it might be hard to judge whether it is still in its native form or not. If you don't have any means of verifying your sample quality, it might be best to discard that batch anyway. But as I said, I don't know about your downstream application so whether it's fine or not really depends on your own judgement.
What buffer is your protein in?
the pH of the buffer in which you are storing your protein may be close to the pI of the protein (may reduce solubility).
Missle on Tue Oct 15 11:56:28 2013 said:
What buffer is your protein in?
Hi,
It is in PBS1X.
mdfenko on Tue Oct 15 13:03:31 2013 said:
the pH of the buffer in which you are storing your protein may be close to the pI of the protein (may reduce solubility).
Thanks for your tip.My protein is in PBS 1X.
map3k on Tue Oct 15 11:19:11 2013 said:
Mariam on Mon Oct 14 17:39:23 2013 said:
Since my protein is not an enzyme, should i check it for the activity?
Well I don't know about your downstream application. If your protein of interest does not have enzymatic activity, it might be hard to judge whether it is still in its native form or not. If you don't have any means of verifying your sample quality, it might be best to discard that batch anyway. But as I said, I don't know about your downstream application so whether it's fine or not really depends on your own judgement.
Thanks for your tips.I want to test its immunogenisity and protection level in mouse.However just the cell immunity response against my protein is important.
another thing, the salt in the buffer may be aiding the precipitation. nacl can be used to precipitate proteins in the same way as ammonium sulfate, especially when the pH is near the pI of the protein.