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protein turn-over


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#1 kli

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Posted 08 May 2014 - 07:14 AM

Hi there,

 

I am in a genetics lab and do not have much experience in biochemistry and please excuse my naive question.

I am planning to look at if my protein A is stabilized by another protein B in cell culture system. So I am going to do a pulse-chase experiment to compare the turn-over rate of protein A in the presence and absence of protein B. I will transfect cells with either A or A+B, radioactively label them, replace the radioactive media and them harvest the cell at different time points after media replacement. Cell lysates will be immunoprecipitated with anti-A antibody. Then the half-life of protein A will be monitored by SDS-PAGE. So my question is: if my hypothesis is true (A is stabilized when B is bound to it), shall I always see more A in the presence of B compared to A alone? Since B will be radioactively labeled and co-immunoprecipitated with anti-A antibody. Thank you very much and look forward to everyone's reply.



#2 bob1

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Posted 08 May 2014 - 12:29 PM

If you assume equal transfection and expression of A in both A alone and A+B, then over time you should see more A in the A+B set of lysates as the stabilization happens.

 

It is entirely possible that you won't get equal transfection, so it may be hard to quantify the differences without an internal standard (another protein you know the half-life of?) or a way of detecting if you have equal transfection.



#3 kli

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Posted 10 May 2014 - 04:47 PM

If you assume equal transfection and expression of A in both A alone and A+B, then over time you should see more A in the A+B set of lysates as the stabilization happens.

 

It is entirely possible that you won't get equal transfection, so it may be hard to quantify the differences without an internal standard (another protein you know the half-life of?) or a way of detecting if you have equal transfection.

Hi bob1, Thank you for your reply. Maybe I did not explain my question well. I was wondering if I will always see more "A" when I co-transfect A+B as long as they bind to each other (but not necessarily A is stablized by B). Or the AB dimer will be broken up when I run a denaturing SDS-PAGE? If denaturing gel does not break up complex, shall I see a higher MW band  (i.e. AB comples) as well as a A band? Does that affect my quantification of A's decay since some A are in the form of AB complex. Thank you.



#4 bob1

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Posted 11 May 2014 - 12:51 PM

It would be unusual to see more A only in a co-transfection, the levels should be about the same as when transfecting independently assuming you are adding equal amounts of the DNAs.

 

The AB dimer should be denatured when running SDS-PAGE - the boiling step with reducing agent (2-mercaptoethanol or DTT) should separate these complexes unless they are very very tightly bound and can re-nature quickly (RNases can do this).  If you want to see your complex you could run native gels.



#5 kli

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Posted 20 May 2014 - 10:50 AM

It would be unusual to see more A only in a co-transfection, the levels should be about the same as when transfecting independently assuming you are adding equal amounts of the DNAs.

 

The AB dimer should be denatured when running SDS-PAGE - the boiling step with reducing agent (2-mercaptoethanol or DTT) should separate these complexes unless they are very very tightly bound and can re-nature quickly (RNases can do this).  If you want to see your complex you could run native gels.

Thank you very much for you reply. One last quick question, what if my B could also activate A transcriptionally. (I guess I could definitely test that by a RT-PCR or western.) But if that is the case, I would see more A protein in A+B than that in A alone right after the "pulse" phase. Can I get around that issue by looking at the turn-over rate/ half-life of A? Thank you.



#6 bob1

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Posted 20 May 2014 - 12:53 PM

If B could activate A transcriptionally, the classic way to test this would be to take the promoter for A and put a reporter gene on it (e.g. luciferase or GFP) and then measure levels of transcript.  However, as you are running off a plasmid, it is unlikely that A will be transcriptionally activated by B unless you have natural promoters, in which case you should also see it with other genes when you co-transfect.

 

I don't think half-life of a protein is any measure of the transcriptional activation, but it is a measure of stabilization.



#7 kli

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Posted 21 May 2014 - 07:45 AM

If B could activate A transcriptionally, the classic way to test this would be to take the promoter for A and put a reporter gene on it (e.g. luciferase or GFP) and then measure levels of transcript.  However, as you are running off a plasmid, it is unlikely that A will be transcriptionally activated by B unless you have natural promoters, in which case you should also see it with other genes when you co-transfect.

 

I don't think half-life of a protein is any measure of the transcriptional activation, but it is a measure of stabilization.

I am not taking half-life of A as a read-out of its transcriptional activity.

I know A does activate B transcriptionally in vivo. My concern is that if B has an A-dependent enhancer within its coding sequence and it works with the basal promoter (hsp70 promoter, for example) in the vector, it might be difficult to interpret my data. I just want to make sure I can conclude A somehow stabilizes B at protein level (not activates B transcriptionally) as long as I see the half-life of B is extended. 



#8 kli

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Posted 21 May 2014 - 07:49 AM

 

If B could activate A transcriptionally, the classic way to test this would be to take the promoter for A and put a reporter gene on it (e.g. luciferase or GFP) and then measure levels of transcript.  However, as you are running off a plasmid, it is unlikely that A will be transcriptionally activated by B unless you have natural promoters, in which case you should also see it with other genes when you co-transfect.

 

I don't think half-life of a protein is any measure of the transcriptional activation, but it is a measure of stabilization.

I am not taking half-life of A as a read-out of its transcriptional activity.

I know A does activate B transcriptionally in vivo. My concern is that if B has an A-dependent enhancer within its coding sequence and it works with the basal promoter (hsp70 promoter, for example) in the vector, it might be difficult to interpret my data. I just want to make sure I can conclude A somehow stabilizes B at protein level (not activates B transcriptionally) as long as I see the half-life of B is extended. 

 

Sorry I wrote is wrong......I am not taking half-life of A as a read-out of its transcriptional activity.

I know B does activate A transcriptionally in vivo. My concern is that if A has an B-dependent enhancer within its coding sequence and it works with the basal promoter (hsp70 promoter, for example) in the vector, it might be difficult to interpret my data. I just want to make sure I can conclude B somehow stabilizes A at protein level (not activates A transcriptionally) as long as I see the half-life of A is extended. 



#9 bob1

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Posted 21 May 2014 - 02:10 PM

So long as you can prove using the half-life that B extends A's half-life, then it should be fine (controls are the key).






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