ubiquitinylated proteins - (May/22/2018 )

Hi,

I'm new to detection of ubquitinylted (Ub) proteins. I'm going to use a kit called Ubi-Qapture-Q kit by Enzo but I'm not sure how I can detect the Ub-protein by western.

This kit isolates ubiquitnlated proteins from cell extract using a high-binding affinity matrix. 

1. When I run the isolated Ub-protein on western gel, I expect to see a smear. The kit says we can use the kit provided Ub-conjugate specific HRP-linked antibody or we can use antibodies to specific protein of interests.  

My question is: isn't it worthwhile to detect the Ub-protein by using antibodies specific to protein of interests.  This way we can tell if our protein of interest is ubquitinated or not?.  What's the purpose of detecting the Ub-protein with the Ub-conjugate specific HRP-linked antibody that comes with the kit?

Also, should we treat our samples with MG132  (proteosome inhibitor) first prior to isolating the Ub-protein by the kit?

Many thanks

A followup to the above question:

If I add MG132, my question would be:

To check if my treatment affects Ub-protein, I have to treat my cells with drug for 48 hrs.  When do I add MG132, a few hours before I lyse the cells or my cells are treated with MG132 and my drug for 48hrs?

1) Yes, you should see a smear if the protein is ubiquitylated. So, if you detect with an antibody against your protein - how do you know that this smear is the result of ubiquitylation and not something like phosphorylation or methylation? The answer is - use an anti-ub antibody and look for co-localization of the stain.

2) Yes - otherwise the Ub is removed by the proteasome.

3) Use the MG-132 for a short time, it will kill the cells within a few hours of addition.

Hi,  thank you very much. What do you mean look for co-localization of the stain?  If I run western using the anti-ub antibody, how do I look for co localization of the stain...sorry I don't understand this part?

By that I meant that you would look for the presence of UB staining in the same region as your protein of interest - you can do this with multi-colour staining on instruments such as the LiCor Odyssey, and the equivalents from other companies. You can also do it by conventional HRP but you need to over-lay images to see anything definite.

Thank you.  On Western blot  I was thinking of running

1.my protein (no treatment),

2.protein treated with MG132 only,

3.protein+drug+Mg132,

4. protein +drug+Mg132 (Ubquitinylated isolated protein).

My target is to observe beta catenin so I would observe to see a smear at the same region (size) of beta catenin in other lanes on my western? sorry for so many questions I'm new to this.

So basically after eluting my protein using the ubquitinated protein kit, I will have to run 2 westerns of the same sample, one to check by anti-ub antibody and second gel by anti-betcatenin antibody?