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Overexpression experiment controls


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#1 miss montana

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Posted 16 October 2009 - 04:42 PM

Hi All,

I want to overexpress 4 different proteins in 293Ts to see if a protein modification occurs when all of them are expressed. What combination of plasmids should I use for my controls (each alone, two of them together, three of them together, etc.)? Right now, my plan includes about 14 different combinations, which seem like a lot. Any input is appreciated!

Thanks.

Edited by miss montana, 16 October 2009 - 04:42 PM.


#2 swanny

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Posted 18 October 2009 - 04:31 PM

Do you have antibodies to all four proteins? Or can you add tags to allow you to see all four at once using different fluoro-Abs? What about the supposed target protein(s)? Can you track what happens to it/them?

What do you aim to control for by your planned controls?
-Successful transformation of the cells? Why not use a selectable marker for each plasmid?
-Expression of the transfected proteins? Fluoro Abs against the proteins or whatever tags you choose to add to them?

A bit more detail of the plan would be great...
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#3 miss montana

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Posted 18 October 2009 - 07:03 PM

Three of the proteins are tagged, and I have a good antibody for the fourth. One of the plasmids is for ubiquitin, the other three are for different proteins. I'm just wondering in what combinations I need to express these proteins. My hypothesis is that the expression of a kinase (one of my proteins) will cause an increase in ubiquitination of another one of the proteins when it's associated with the third protein that I am expressing. I won't be looking at any endogenous proteins to start.

I think my main controls will have to show that all three of the proteins are required for ubiquitination (if this is actually true) to show that the kinase is causing the modification. Therefore, I'd do kinase + 1 protein, kinase + 2 proteins, 2 proteins without kinase, kinase + all three proteins, all three proteins without kinase. Or is this overkill? Thanks!

Do you have antibodies to all four proteins? Or can you add tags to allow you to see all four at once using different fluoro-Abs? What about the supposed target protein(s)? Can you track what happens to it/them?

What do you aim to control for by your planned controls?
-Successful transformation of the cells? Why not use a selectable marker for each plasmid?
-Expression of the transfected proteins? Fluoro Abs against the proteins or whatever tags you choose to add to them?

A bit more detail of the plan would be great...






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