i agree with irene:denaturing the membrane before blocking is very helpful- i use wet-blotting, (NC or VDF), denaturate membranes for 30min/4°C (6M guanidinium chloride in 20mM Tris pH 7.5, 1mM PMSF, 10mM NEM, 5mM ß-mercaptoethanol-this would be 8µl for 25ml; Penengo L et al. Cell 2006. FK2 and P4D10 work fine...

Personally, I use this one: http://www.dako.com/dist/ar38/p106260/prod_products.htm?setCountry=true&purl=ar38/p106260/prod_products.htm?undefined&submit=Accept%20country

I would recommend one from Abcam, they have many different ones all rated for different assays and species types. We use a human one for western blotting with very nice success.

With that said, we never perform just straight westerns with a ubiquitin antibody as they are always large smears (due to so many proteins being conjugated). I would recommend you perform an IP first for the IRS-2 protein, then western for ubiquitin. If there is not a IP grade antibody for IRS-2, then pull down with an IP grade ubiquitin antibody and blot for IRS-2.

Good luck.

~Adam

I have used this one: http://www.scbt.com/datasheet-8017-ub-p4d1-antibody.html , with quite good result. I treat the blot with a denaturing solution prior blocking.

Good luck!

The FK2 antibody works great in both human and rat samples (I haven't tried mouse) by western blot and immunofluorescence. This antibody should recognize the ubiquitin modifications (both mono and poly) on IRS-2. The DAKO antibody that others have suggested is also a good option.

http://www.enzolifesciences.com/BML-PW8810/mono-and-polyubiquitinylated-conjugates-mab-fk2/

Hi Nagaraj,

I agree with Adam. I recommend that you Ip your protein then Wb. In my previous project I used monoclonal ubiquitin antibodies from (Zymed) and it worked very well.

Good luck

i agree with irene:denaturing the membrane before blocking is very helpful- i use wet-blotting, (NC or VDF), denaturate membranes for 30min/4°C (6M guanidinium chloride in 20mM Tris pH 7.5, 1mM PMSF, 10mM NEM, 5mM ß-mercaptoethanol-this would be 8µl for 25ml; Penengo L et al. Cell 2006. FK2 and P4D10 work fine...

The best antibody for Western blot (as well as for IHC):

http://www.lifesensors.com/pdf/VU101_VU-1Antibody_datasheet_032113.pdf;

http://www.lifesensors.com/ubiquitin-research-tools/ubiquitin-ubl-pathway-antibodies/antibodies/vu-1-ubiquitin-mouse-monoclonal-antibody/p322.

It recognizes mono- and polyubiquitylated proteins as well as free ubiquitin (important marker). It works better than FK2 if run side by side.

Honestly, none are very good. Best method is to use an expression construct with an ubiquitin insert, and immunoprecipitate your protein of interest and then immunoblot for the tagged ubiquitin construct. This has given me the best results in the past.

I think there's a bunch out there, and it depends if you need to look at ubiquitin alone or ubiquinated proteins. You can always go to Abcam but they are very pricey and don't make the products themselves generally. Some good alternatives would be found at http://stressmarq.com/Products/Antibodies/SPC-119B.aspx that can see both the free and ubiquinated proteins, for free ubiquitin alone http://stressmarq.com/Products/Antibodies/SMC-171B.aspx or http://stressmarq.com/Products/Antibodies/SMC-160B.aspx work well for human/mouse. Hope this helps.

I look for an anti-Ub to be used in s.cerevisiae to detect Rpb1-Ub. I have tried the P4D1 from Santa Cruz and it does not work.

Hi Francisco, I agree with you that anti-Ub from santa cruz is to bad for Ub detection. Try monoclonal ubiquitin antibodies from (Zymed) work well..have been using that for years.

Good luck

Does it work well with S. cerevisiae? Do you know the exact reference or name?

thanks

I need to do a similar kind of work. The aim is to pull down all the ubiquitin conjugated proteins in infected cell line. Can anyone suggest me which MAb should i use. How to differentiate between the free ubiquitin and conjugated uboquitin ?

http://www.scbt.com/datasheet-8017-ub-p4d1-antibody.html

1 to 1000 dilution ON at the cold room

ENZO Lifesciences (http://www.enzolifesciences.com/) has a great number of ubiquitin antibodies. Many of them work well depending on what kind of ubiquitin linkage you are trying to detect. They have a polyubiquitin antibody that detects K29-, K48-, and K63-linked mono- and polyubiquitinylated proteins quite well.

VU-1 (http://www.lifesensors.com/ubiquitin-research-tools/anti-ubiquitin-detection-and-isolation-tools/ubiquitin-antibodies/vu-1-ubiquitin-mouse-monoclonal-antibody/p322#.VfaTD5f-ltE) is most sensitive to my opinion. I block with 2% BSA and use the antibody 1:1000 O/N at the coldroom. 

What concentration do people use the DAKO Ubiquitin antibody at for western?