I'm a greenhand doing Western blot. When I was using anti-UCP1 antibody from rabbit (produced by Sigma) to detect UCP1 in total protein extracted from differentiated brown adipocytes (I wrongly wrote mice interscapular brown adipose tissue (iBAT), corrected now), aside of the UCP1 band (~32 kDa), there was always a non-specific band around 50 kDa. I wonder if that was a common non-specific band appear in such experiment or it was due to the antibody we use? Could that band be something like IgG heavy chain (for some sellers say that it could be, but their protein samples were directly from tissue rather than cells), or it was more likely to be other protein (and how could I prove it)?
Thanks a lot in advance!
That depends highly on how you are detecting UCP-1 on your western blot. You said you are using a rabbit antibody to UCP-1. Is that antibody conjugated to your detection system [i.e., an enzyme like alkaline phosphatase or luciferase] or are you using a conjugated anti-rabbit antibody to detect the antibody:UCP complex? If so, if that antibody is polyclonal [i.e., goat anti-rabbit] it might cross-react with mouse Ig heavy chain.
You could block that by adding either purified mouse IgG or normal mouse serum to your detection reagent which would bind to the cross-reactive antibodies and block binding to the mouse Ig heavy chain, which should take care of your problem.
What I have used in the past was 10 ug/ml purified mouse IgG [1:100 dilution of a 1 mg/ml solution] or 1:200 dilution of normal mouse serum and filter through a 0.2 um Tuffryn syringe filter [Acrodisc by Pall Corporation]. These filters are very low volume retention, which saves on expensive reagents.
Gary Lee Gilmore
Hi Gary, I am intrigued as to how you go about avoiding appearance of both heavy and light chain on western blots when using mouse monoclonal Abs on mouse lysates.
You mentioned the use of both Mouse IgG and/or mouse serum.
Is that added to the membrane whilst in primary antibody or at some other stage (Secondary). Would this avoid the detection of the IgG h + L on the WB after HRP based detection?
I would be curious to know! Thanks v much.
Satish
You are asking something different than what I was responding to. If you are using a mouse monoclonal antibody to detect your antigen, absorbing anti-mouse reactivity in your secondary antibody is NOT what you want to do. But if you are using a non-mouse primary antibody [in the current case, rabbit] to target your antigen and a polyclonal antibody from a different species that may cross-react with mouse IgG in your sample, you can block that binding [the immunologic term is 'absorb'] by adding either purified mouse IgG or normal mouse serum. It has been a long time since I've done this but my memory is we used a 1:4 dilution normal mouse serum. That was added to the detection antibody at 1:50 as I described, and filtered with a Tuffyn membrane micro filter prior to addition to the detection reagent. I've also used purified mouse IgG at a final concentration of 10 ug/mL.
Check this brief https://www.cygnustechnologies.com/media/productattach/n/o/non-specific_binding_in_westerm_blot_10.16.18.pdf
Also check https://www.bioradiations.com/western-blot-detection-without-interfering-iggs-a-mere-fantasy-or-a-real-possibility/
Check on this paper
https://www.bu.edu/picf/files/2019/05/Azure-Western-Blotting-Guidebook.pdf
Hello, I am interested to know how you ended up resolving this issue with UCP1 antibody. Thanks!
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