Hi Micheal,

With the myc pulldown you are purifying both the non-modified protein and the ubiquitin modified one. Since the fraction of a given protein that is ubiquitinated is usually very small it is usually very difficult to detect by western blot, unless you enriched that ubiquitinated fraction.

With myc antibody the ubiquitin modified protein might remain undetected because you are mostly detecting the non-modified form. Using anti-ubiquitin antibody might help(FK2 antibody work very well for us). However, as Huibing Wang and Hideyo Yasuda have already suggested, I would also recommend that you transfect these cells also with a tagged-version of ubiquitin. This will allow you do an IP with the tagged-ubiquitin (so you enriched the ubiquitinated material) and then do a western blot to you protein. Since in the IP you have purified only proteins bound to ubiquitin in the western blot you should see a band 10 kDa higher (or more, depending on how many ubiquitins attached it has) than the band seen in the inputs. Alternatively, you can do an IP with the tagged of your protein and then use the tagged of the ubiquitin to detect the ubiquitinated fraction, which will be easier as you are overexpressing ubiquitin. We have done both approaches and it worked fine for us. Once you confirm that your protein is ubiquitinated in this condition you can then try to see the endogenous ubiquitination levels without overexpressing any ubiquitin. 

Regarding the MG132 or Bortezomid, in my experince this treatment only worked if the protein is degraded through the proteasome.

I hope it helps

Good luck!!!

Juanma

How much protein did you use? That can be one point to look at. Use total protein >100ug for IP. Is the single band the band from your protein with myc tag? Can you have mono-ubiquitination? Ubiquitination is a transient condition...even with MG132 you may not detect the ladder...you have to start from a lot of material!

Thank you very much.

I used protein from two 10cm dishes for IP, which should be over 100ug. The single band should be a ladder band. The size is about similar to tagged protein. There is no report about ubiquitination of my protein yet.   I may try different Ab instead of myc first to see what  I will get.  Is there any suggestion should I care about? 

Hi Micheal,

So you don't know if your protein is ubiquitinated or not. I agree with Hideyo Yasuda about using poly-ub ab to detect if it is ubiquitinated, but I would also suggest you to test degradation assays in the presence or absence of MG132 to check if the proeasome affects the stability of your protein (do you have your protein purified?). If the proteasome affects the stability of your protein then you may try to see poly-ubq...

I hope it helps...

Best regards

@Huibing Wang  Thank you!　 I did see polyub before- I think...

＠Hideyo Yasuda　ｔｈａｎｋ　ｙｏｕ　ｖｅｒｙ　ｍｕｃｈ．　Ｍｙ　ｌａｂｅｍａｔｅ　ｕｓｅ　ｓａｍｅ　Ａｂ　ａｎｄ　ｇｅｔ　ｌａｄｄｅｒ　ｂａｎｄ．　Ｔｈａｎｋ　ｙｏｕ　ｉｆ　ｙｏｕ　ｃｏｕｌｄ　ｓｈａｒｅ　ｙｏｕｒ　ｐｒｏｃｅｄｕｒｅ！

＠Tiago Lourenço　　Ｔｈａｎｋ　ｙｏｕ　ａｇａｉｎ！Ａｃｔｕａｌｌｙ，　Ｉ　ｄｉｄ　ＭＧ１３２　ｔｒｅａｔ　ｓｅｖｅｒａｌ　ｔｉｍｅｓ．　Ｓｏｍｅｔｉｍｅｓ　Ｉｔ　ｄｉｄ　ｉｎｃｒｅａｓｅｄ　ｗｉｔｈ　ＭＧ１３２，　ｂｕｔ　ｓｏｍｅｔｉｍｅｓ　ｄｉｄ　ｎｏｔ．　

@Huibing Wang Thank you. I havent  done that yet. I read some articles that they did endogenous  ub. 

Thank you very much for your kind reply, Hideyo! I will do Bortezomb treatment first  to see what I could get. 

Best wishes for all you guys!

Hi Micheal,

With the myc pulldown you are purifying both the non-modified protein and the ubiquitin modified one. Since the fraction of a given protein that is ubiquitinated is usually very small it is usually very difficult to detect by western blot, unless you enriched that ubiquitinated fraction.

With myc antibody the ubiquitin modified protein might remain undetected because you are mostly detecting the non-modified form. Using anti-ubiquitin antibody might help(FK2 antibody work very well for us). However, as Huibing Wang and Hideyo Yasuda have already suggested, I would also recommend that you transfect these cells also with a tagged-version of ubiquitin. This will allow you do an IP with the tagged-ubiquitin (so you enriched the ubiquitinated material) and then do a western blot to you protein. Since in the IP you have purified only proteins bound to ubiquitin in the western blot you should see a band 10 kDa higher (or more, depending on how many ubiquitins attached it has) than the band seen in the inputs. Alternatively, you can do an IP with the tagged of your protein and then use the tagged of the ubiquitin to detect the ubiquitinated fraction, which will be easier as you are overexpressing ubiquitin. We have done both approaches and it worked fine for us. Once you confirm that your protein is ubiquitinated in this condition you can then try to see the endogenous ubiquitination levels without overexpressing any ubiquitin. 

Regarding the MG132 or Bortezomid, in my experince this treatment only worked if the protein is degraded through the proteasome.

I hope it helps

Good luck!!!

Juanma

Hi Juanma,

Thank you very much for your patient ！ I thought detecting endogenous ubiquitination is more convince. I would try MG132 and Bort. again and then do transfection.

hello,

Yah, we usually use MG132 for ub detection. Also, if you are getting single band at the size of protein of your interest it might be monoubiquitination. 

Hi Swapnil, Thank you! I will do transfection first.

You could put NEM or other alkylating agent in the lysisbuffer and washing buffers. This reacts with free thiols of cysteines, such as those of the active site in DUBs, and prevents deubiquitination post-lysis. I have seen deubiquitination post-lysis, even after immunopreciptation of the target protein.

 @Juanma Ramirez，thank you very much. I would do transfection first. 

@Juanma Ramirez Hi, do you pre-heat the lysate samples before do the IP? It seems to be useful to break binding between your protein of interest and others.

@Mara Esposito ·Does that help to bind interest protein? Could you tell  the detail?