I'm working on the ubiquitination of a protein and to understand the type of ubiquitination, I used MG-132 and lactacystin proteasome inhibitors. Interestingly, I have a huge decrease of my ubiquitinated protein when I put the drug compare to the control.
MG-132 is the inhibitor against the proteasome 26S and used for the treatment for multiple myeloma in the clinical settings. Actually, I have the similar experimental result, when I treated human melanoma cells with MG-132 for long time course up to 24hr. Too much concentration or too long time treatment might cause the paradoxical output, I mean, the half-life time of the protein becomes longer!!! I still do not know the exact reason, but I strongly recommend you to treat cells of interest with MG-132 for the short time course such as 0.5,1,3,6 hr.
It is still there! You have to run the insoluble fraction that is the protein that has been heavily ubiquitinated. Ubiquitinated proteins tend to form aggregates. If you solubilized with RIPA or TX 1% you still have protein that will form an insoluble fraction which will be located in the pellet. Solubilize the pellet with SDS 2% and then dilute it. The protein will show up.
What system are you working in?
--> Etienne Cartier_ I understand what you mean, but why would that not be the case when he doesn't add MG132? He says he can see the ubiquitinated form of the protein under normal conditions, but why with the addition of MG132 would he all of a sudden have a problem with solubility?
I'm working with HEK293T cells, I transfect Ubiquitin and my protein of interest and 2 days after, I treat the cells with 10µM of MG132 during 6h. As control I add DMSO.
After that I do an immunoprecipitation against a tag (in N-terminal part of my protein) and I do an immunodetection against the tag of the transfected ubiquitin.
Clearly there is an decrease of the "ubiquitin signal" in the MG132 conditions compare to the control.
Maybe It's more clear :)
Do you see an increase in polyubiquitinilated proteins in the mg-132 sample compared to the control in your input?
Yes, frist I do a western blot to detect in one membrane my protein of interest and in another membrane I detect the transfected ubiquitin trough its tag and by eye, I see a difference in the intensity of the smear compare to the control and the MG132.
More I see also a difference in the EGFR who shows an augmentation of the poly-ubiquitinated EGFR :)
I am not quiet sure I understand it right. In your input samples under treatment with MG-132 you see an increase in polyubiquitinated proteins, as well as of your protein of interest. But when you IP the protein of intrest you see a decrease in ubiquitin forms under MG-132 treatment?
As you protocol seems to be working for the EGFR, I would assume that you probably don't have residual DUB activity in the samples which may could have lead to ubiquitin removal during the IP procedure.
Is it possible that the N-terminal tag may masked non-classical ubiquitinylation at the N-terminus? Did you pull down endogenous protein and look at ubiquitin? Did you try inhibition of protein synthesis?
Are you comparing a control IP with your MG132/lactacystin IP? Or are you looking at lysates vs. your MG132/lactacystin IPs? Are you controlling for DUB activities anywhere (as mentioned above)? Could try including NEM, iodoacetamide and some EDTA in your IP lysis buffer...
As control for my IP, I use the same condition (protein of interest and ubiquitin) without treatment of MG-132, just the solvent (DMSO).
Effectively, I should try an inhibitor of DUBs but at the beginning I though that andogenous DUBs should have no much effects.
I looked for different inhibitor :
The NEM is the most used but there are side effects? Because this inhibitor is not specific of the DUBs.
The Ub-aldehyde is more specific but for me there will be a competition between Ub and this Ub-aldehyde for the substrate; no?
Normally use NEM only in a lysis buffer. There shouldn't be any competition between Ub and Ub-al for ubiquitylation of the substrate protein. Pretty sure Ub-al can't be used by E1/E2/E3 and only inhibits DUBs.
Question for Go J Yoshida, or anyone else used MG-132 for 24 hours.
You wrote you try to use MG-132, which concentration did you use? are the cells look good after the treatment?
did you check the insoluble fractions?? Ubq proteins tend to get quite insoluble...
Without MG132, I see clear polyubiquitination bands so I think it's not insoluble problem issue .
I guess some DUB is accumulated by MG132.
yes, but when you inhibit proteasome there is a ton of Ubq. Check Ron Kopito's paper (90s)...is a Cell...
Cell. 1995 Oct 6;83(1):121-7.
Degradation of CFTR by the ubiquitin-proteasome pathway.
Ward CL1, Omura S, Kopito RR.
Of course. There are tons of polyub chains in whole lysate but after IP with specific target, most ub chains on the target were gone. When compare between with / without MG132 I see more dramatic accuration of ub signals in no MG132 condition.
check the insolubles. Otherwise, does not make any sense to me...
I will try that. Thank you for your comment.
Btw, I did IP after denaturing with 2% SDS. Do you think there are still insoluble proteins? I think 2% SDS makes most proteins soluble.
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