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ubiquitinylated proteins


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#1 SF_HK

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Posted 21 May 2018 - 11:48 PM

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



#2 SF_HK

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Posted 22 May 2018 - 12:03 AM

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?



#3 bob1

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Posted 22 May 2018 - 01:15 PM

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.



#4 SF_HK

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Posted 22 May 2018 - 03:38 PM

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?



#5 bob1

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Posted 23 May 2018 - 06:12 AM

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.



#6 SF_HK

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Posted 28 May 2018 - 11:22 PM

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.



#7 SF_HK

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Posted 28 May 2018 - 11:45 PM

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?






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