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How to look at protein stability?


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#1 rocketfan86

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Posted 05 May 2010 - 07:53 AM

I am studying a protein that has specific ubiquitin-lysine residues.

How are some ways to determine if mono-ubiquitination of these residues stabilizes the protein and increases half-life or vice versa.

Someone was telling me about pulse-chase but wouldn't that just label all newly synthesizes proteins?

Is tagging the protein with GFP enough to monitor half-life/localization during normal and stressed conditions?

Any help is appreciated. Thanks.

#2 Another Jake

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Posted 05 May 2010 - 10:37 AM

Pulse chase is still the way to go. Yes, it will only label protein as it is made. That is why you want to do it. When you add the 35S-Met, you set the zero time point. Do a very, very short pulse... on the order of 5 or so minutes (try a few times). At 5, 10, 20, 40min (or whatever, up to you) time points following the labeling, move the plates to ice, lyse them, and collect the samples as quickly as you can. Do an IP, gel, etc. Since you've only labeled protein that was made during that 5 minute label, you will be able to watch that particular population degrade; from that data you can easily calculate a half-life. GFP would not be the way to go. The tag can have an effect on the stability of the protein. That means tons of controls. Do the pulse-chase.
Compare the wild-type protein to a clone which has point mutations at the ubiquitination sites.
There are also antibodies out there that only recognize mono-ubiquitinated proteins, but they are notoriously crappy antibodies.

#3 rocketfan86

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Posted 05 May 2010 - 01:33 PM

Pulse chase is still the way to go. Yes, it will only label protein as it is made. That is why you want to do it. When you add the 35S-Met, you set the zero time point. Do a very, very short pulse... on the order of 5 or so minutes (try a few times). At 5, 10, 20, 40min (or whatever, up to you) time points following the labeling, move the plates to ice, lyse them, and collect the samples as quickly as you can. Do an IP, gel, etc. Since you've only labeled protein that was made during that 5 minute label, you will be able to watch that particular population degrade; from that data you can easily calculate a half-life. GFP would not be the way to go. The tag can have an effect on the stability of the protein. That means tons of controls. Do the pulse-chase.
Compare the wild-type protein to a clone which has point mutations at the ubiquitination sites.
There are also antibodies out there that only recognize mono-ubiquitinated proteins, but they are notoriously crappy antibodies.


Thanks for the reply. How do you identify your protein of interest? Would you IP and blot it at these different time points?

#4 Another Jake

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Posted 05 May 2010 - 01:52 PM

IP with the antibody against your protein of interest. The pulse-chase is done with radioactive S35-methionine (look at publications for the protocol). The IP will pull down the protein you want... and unfortunately some other stuff you don't, but mostly the one you want. Dry the gel onto whatman with a gel-dryer, put film on it for 24 hours first, then longer if needed. That should be a good start. If you don't have a gel-dryer, you can transfer the protein to a membrane... the assay will still work, but you lose sample in the transfer, inevitably. If you have phosforimager plates and the phosphorimager machine itself, that works better for quantitation... and gives you a better signal faster.




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