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Use of urea in solubilizing protein and its aftermath

urea inclusion bodies protein purification dialysis

Best Answer mdfenko, 20 June 2016 - 07:20 AM

1. urea denatures the protein. if it is an enzyme then it will not exhibit enzymatic activity. the denaturing caused by urea can be reversed (the protein can be renatured or refolded) by removing the urea but it will not return all of the protein to the native state. antibodies to the native protein may not recognize the denatured protein.

 

2. it should not affect a western blot if it is prepared from an sds gel.

 

3. if you don't remove urea slowly then you will most likely misfold most of the protein. by reducing the urea slowly you have a better chance of correctly refolding the protein.

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#1 Meg P. Anula

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Posted 19 June 2016 - 06:12 AM

Hi, so most of my protein that I wanted to express by E.coli BL21(DE3) is in inclusion bodies and hence I used 8M of urea in my binding and elution buffer when I purified the protein with Histrap column.

 

My question is :

 

1. what's the bad consequences of using urea? I sure there must be some big disadvantages, otherwise people would just include urea in all buffers without testing the protein solubilization prior. Is it because it's a denaturant hence the protein will be in denatured form? And what will it affect?

 

2. Will my protein in elution buffer containing urea affect the western blot results later?

 

3. And to eliminate urea from protein, why do people do dialysis in descending order or urea instead directly in buffer without urea at all?

 

 

I appreciate your help. Thanks in advance.

 

 



#2 mdfenko

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Posted 20 June 2016 - 07:20 AM   Best Answer

1. urea denatures the protein. if it is an enzyme then it will not exhibit enzymatic activity. the denaturing caused by urea can be reversed (the protein can be renatured or refolded) by removing the urea but it will not return all of the protein to the native state. antibodies to the native protein may not recognize the denatured protein.

 

2. it should not affect a western blot if it is prepared from an sds gel.

 

3. if you don't remove urea slowly then you will most likely misfold most of the protein. by reducing the urea slowly you have a better chance of correctly refolding the protein.


talent does what it can
genius does what it must
i do what i get paid to do

#3 Meg P. Anula

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Posted 20 June 2016 - 07:58 PM

1. urea denatures the protein. if it is an enzyme then it will not exhibit enzymatic activity. the denaturing caused by urea can be reversed (the protein can be renatured or refolded) by removing the urea but it will not return all of the protein to the native state. antibodies to the native protein may not recognize the denatured protein.

 

2. it should not affect a western blot if it is prepared from an sds gel.

 

3. if you don't remove urea slowly then you will most likely misfold most of the protein. by reducing the urea slowly you have a better chance of correctly refolding the protein.

 

 

Thanks so much for the explanation. I have another question, how does antibodies recognized a denatured protein? Denatured means the protein unfolded back to primary structure right? Doesn't antibodies apply lock-and-key theory as well? 



#4 mdfenko

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Posted 21 June 2016 - 04:31 AM

some antibodies will develop against the 3 dimensional structure but others, especially those developed against short peptides, are sequence specific. denaturing will better expose these sequences. it all depends on the epitope to which the antibody is derived.


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#5 Meg P. Anula

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Posted 21 June 2016 - 06:53 AM

I see. I used to run SDS page/ western blot and my antibodies manage to detect the protein. Since SDS itself is denaturant, I supposed it means the antibodies I used can recognize the protein in denatured form, am I right? 



#6 mdfenko

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Posted 22 June 2016 - 03:43 AM

you are correct


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