Quantification of Ubiquitination

[Thitus]

Hi,

I would like to know if there is a method quantify/assess the degree of ubiquitination of a protein of interest in tissue lysate?
My idea was to do a co-immunoprecipitation of polyubiquitin and my protein but I am afraid this is not quantitative enough. What do you think?
Or is there a method to quantify the proteasomal degradation of a protein x in vitro?

I have no experience with this area and would greatly appreciate your help!

[admin]

Hello, welcome to the site!
you can either try measuring Ubiquitination indirectly by antibodies (western blot, qualitative western or anti-ubiquitin immunocytochemistry) or maybe even try direct by radioactivity or similar non-radioactive label

[Thitus]

Hi,

Maybe I wasn't concrete enough with my question. I found via Western Blot that protein X is more abundant in brain tissue lysate of sick mice as compared to controls. Surprisingly, my Rt-PCR experiments do not show a difference in mRNA levels.

That made me think of less degradation being a potential reason (assuming my Rt-PCR worked correctly!)

I speculate that is could be due to:

1. less proteasome levels
2. decreased proteasome activity
3. decreased ubiquitination of protein X


I do not know how I would check for 3. I don't think Western Blot or Immuno is the way to go, as this would give me the total amount of ubiquitination in my tissue.

How can I assess and compare the level of ubiquitination specifically of protein X in my mice? I was thinking of immunoprecipitation, but I do not know whether it would be quantitative enough.

I would immunoprecipitate protein x and blot for ubiquitin.


What do you think?


Thank you for your input!:-)