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Protein precipitation by storage

recombinant protein precipitation storage

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11 replies to this topic

#1 Mariam

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Posted 13 October 2013 - 09:42 AM

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.sad.png


Edited by Mariam, 13 October 2013 - 10:59 AM.


#2 map3k

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Posted 14 October 2013 - 01:35 AM

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



#3 Mariam

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Posted 14 October 2013 - 09:39 AM

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?


Edited by Mariam, 14 October 2013 - 09:43 AM.


#4 map3k

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Posted 15 October 2013 - 03:19 AM

 

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.


Edited by map3k, 15 October 2013 - 03:19 AM.


#5 Missle

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Posted 15 October 2013 - 03:56 AM

What buffer is your protein in?



#6 mdfenko

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Posted 15 October 2013 - 05:03 AM

the pH of the buffer in which you are storing your protein may be close to the pI of the protein (may reduce solubility).


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#7 Mariam

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Posted 15 October 2013 - 08:53 AM

What buffer is your protein in?

Hi,

It is in PBS1X.



#8 Mariam

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Posted 15 October 2013 - 08:55 AM

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.



#9 Mariam

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Posted 15 October 2013 - 08:59 AM

 

 

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.



#10 mdfenko

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Posted 16 October 2013 - 03:49 AM

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.


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#11 labtastic

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Posted 16 October 2013 - 04:42 AM

Run some of your re-dissolved protein over a gel-filtration column.

 

If you get a single, monodisperse peak eluting at the expected molecular weight, then chances are your protein is perfectly fine. If you see some aggregation and polydispersity in the protein sample that by SDS-PAGE is a clean, single band, then I would hesitate to use it for any real application.

 

If you don't have a gel filtration setup in your lab, then simple alternative is to run Native-PAGE on the sample. If the expected pI of your protein is <7, then all you have to do is run the gel like a normal SDS-PAGE gel except leave out all SDS (in your sample buffer, running buffer and gel itself). Stain with coomassie and if you get a nice tight band then chances are your protein is good. If it comes out as aggregates, oligomers or an uninterpretable smear, then it's no good.



#12 Mariam

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Posted 16 October 2013 - 07:38 AM

Run some of your re-dissolved protein over a gel-filtration column.

 

If you get a single, monodisperse peak eluting at the expected molecular weight, then chances are your protein is perfectly fine. If you see some aggregation and polydispersity in the protein sample that by SDS-PAGE is a clean, single band, then I would hesitate to use it for any real application.

 

If you don't have a gel filtration setup in your lab, then simple alternative is to run Native-PAGE on the sample. If the expected pI of your protein is <7, then all you have to do is run the gel like a normal SDS-PAGE gel except leave out all SDS (in your sample buffer, running buffer and gel itself). Stain with coomassie and if you get a nice tight band then chances are your protein is good. If it comes out as aggregates, oligomers or an uninterpretable smear, then it's no good.

Thank you vary much for your valuable tips.I will try it.







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