Ubiquitination - (Nov/08/2005 )

Hi there!

Does anyone know anything about ubiquitinated proteins? I am looking for ubiquitination on my protein that is not signalled for degardation. I carried out an IP and immunoblot for ubiquitin and what I got was a smear. Is it an artefact or should I incubate my cells with MG132 for lesser time? Normally i incubate it for 24hr pre-harvest at10um. How should i know my protein is signalled for degradation?

Any suggestion would be good. Thanks so much!

Weini

---

Hi

Your protein is not targeted for degradation? that´s what you said?
proteins can be degraded basically by to systems, proteosome ubiquitination system or by the lysosomes. If your protein is degraded by the proteasome then it has to be targeted for ubiquitination.

is not the first time I see in articles, western blots where you see the band of the protein expected size and above that a smear, corresponding to the polyubiquitinated protein. You check out in literature.
But do you see your normal band or just the smear?

---

usually i keep the cells in 10um MG132 for no longer than 8h. cells dont like it, do you see if anything is hapening to them?
do you have a control with MG132 no treated cells? do you see an increase in ubiquitinylation upon MG132 treatment?if you see an increase in ubiquitinylation level after MG132 treatment, i think that could be an indication that your protein goes through proteasome.. i think the smear itself could mean its either polyubiquitin binding (that would count for degradation) or maybe you have monoubiquitinylation on multiple lysins (which means different function). how do you know that your protein is not signalled for degradation?

---

hello,

if i understand correctly, MG132 is a proteasome inhibitor, not an inhibitor of ubiquitylation. therefore, you would expect to see your protein as a poly-ubiquitin conjugate after 24 hr, and that would probably look like smear on a western blot.

i do have a question for you though...

did you perform your IP using antibodies to your protein, or to ubiquitin (it's not entirely clear from your question)? if you can, try doing a negative control (no MG132, or a 0hr time point) in parallel with your MG132 samples. if your protein is subject to routine turnover via the ubiquitin/26S proteasome, you'd expect to see less Ub-conjugated species (as they'd be degraded and not just "stack up" like they would in the presence of a proteasome inhibitor) in your 0hr or no MG132 controls. the same should be true if you performed an IP using anti-Ub Abs and probed your immunoblot w/ Abs to your protein of interest.

long story short, the "smear" you see is probably (as another reply to your post already stated) poly-Ub conjugated forms of your protein- just be fussy about your controls, and it should sort itself out.

if you have further question, feel free to email me.

-jon

jonmike.reed@gmail.com

---