ubiquitin antibody from cell signaling(3933), source rabbit,1:1000 dilution
It's unlikely to work, as ubiquitinated proteins tend to be rapidly degraded. If you're looking at ubiquitination in general, I recommend treating cells withe the protease inhibitor MG132. If you're trying to look at a specific protein, it's best to isolate it. The best method is to His-tag the protein of interest and purify it over a nickel column under denaturing conditions.
To study the ubiquitylation of proteins, it is very important to handle your cells and samples properly. If you can not treat your cells with proteasome inhibitors MG115 or MG132 for some reason, then you must keep the cell lysis on ice all the time otherwise I recommend that you treat the cells with the Proteasome and protease inhibitors. Immune-precipitate your protein of interest and then continue with wb. Cell signaling has the best Ubiguitin antibodies according to my experience and works very well. Of course the protein of interest plays an important role. How much there is in the cell, how rapidly degraded and the protein's size is around 50-55 KDa will complicate detection since the Ig heavy chain impairs detection. I agree with Jillian that His-tag the protein of interest and purify it over a nickel column under denaturing conditions but this is not always is good solution due to you have over express level of proteins and would react differently than endogenic protein.
good luck
No you will not be able to detect the ubiquinated proteins without their enrichment in the lysate, especially if you are looking for a particular ubiquinated protein.
Hi !
I worked on ubiquitination for a few years, and I never "enriched" the lysate to detect the global ubiquitination in the cell, neither using any DUB or proteasome inhibitor (by the way, ubiquitination is absolutely not systematically related to proteasomal degradation but has many other functions!).
If you are interested in the ubiquitination level of a particular protein, you have to (immuno)precipitate it.
I used to lyse a 100mm dish of HEK293T in 1mL of lysis buffer and use 15-20uL for the blotting. To detect the ubiquitinated proteins, I used the FK2 antibody from Enzo.
Another possibility, is to use a HA-tagged ubiquitin expressing plasmid...
Good luck.
Anne
I can only confirm that it is not required to treat cells to detect ubiquitination in general. Looking for a specific protein, depending on its abundance, it might be necessary to use MG-132 or a more specific reagent that blocks the particular ubiquitination/degradation pathway at a specific point to stabilize the substrate.
LYSIS: Try to be as quick as possible, always on ice… If you can, directly use hot SDS sample buffer for lysis… by far the quickest lysis! I did time course studies and looked at HIF1alpha ubiquitination and degradation… HIF1alpha for example can be a pain in the neck, its turned over very quickly, t1/2 is estimated to be about 5 min..
Good luck!
Kurt
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