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Protein fragment is alwasy degraded post transfection 48 hrs

protein degraded

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#1 lilin_hust

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Posted 09 March 2014 - 01:01 PM

Hi, I am meeting problems with my IP. I have a protein and, I get 3 fragment of this protein expressed, and trying to see which one is responsible for the binding.

 

However, while the C-terminal and N-terminal fragment is Okay to express, I can't see any signal for the middle fragment, I think it was because the protein expressed was degraded in vivo before I extract my protein.

 

So will it be any better if I collect my cells earlier like 24 hrs rather than 48 hrs post-transfection??

 

Anyone ever experience this? Thanks!!!



#2 jerryshelly1

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Posted 10 March 2014 - 01:33 PM

So....You are transfecting a cell that is expressing three different versions of your protein? Are you sure you are not losing the cell during the protein extraction phase? Have you tried increasing the amount of protease inhibitors you are using? What about your lysis conditions...Are they to stringent for the protein, which is causing degradation? Even if your protein was being degraded, you would see something on your blot (additional bands, smears, etc.).

 

Do you have a picture of your blot you can post?

 

Can you also elaborate on your protein you are trying to express. Are you expressing three different versions of your protein or is the three fragments the result of a modification?



#3 lilin_hust

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Posted 14 March 2014 - 09:19 PM

So....You are transfecting a cell that is expressing three different versions of your protein? Are you sure you are not losing the cell during the protein extraction phase? Have you tried increasing the amount of protease inhibitors you are using? What about your lysis conditions...Are they to stringent for the protein, which is causing degradation? Even if your protein was being degraded, you would see something on your blot (additional bands, smears, etc.).

 

Do you have a picture of your blot you can post?

 

Can you also elaborate on your protein you are trying to express. Are you expressing three different versions of your protein or is the three fragments the result of a modification?

Hi, Jerry, because two of the plasmids are expressed quite well (namely the N terminal and C terminal fragment), but the middle part I can't detect any. So i am thinking that the middle part is expressed, but is degraded soon by the cell machinery such as lysosome.

 

So I want to know how I can make the expressed protein fragment to be degraded to the least content that I shall be able to detect at least some signal.(for my IP to determine which part is responsible for the binding)






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