We are using UCP-1 antibody on samples of BAT which should show up around 32-35 kda. While some of our samples have the bands at 32 kda where we want them, we are consistently getting a band around the 26 kda mark, even with our negative controls. Basically every sample is showing this and are exactly in the same spot. Has anyone had issues with double bands with the ab10983 antibody? Perhaps it is something I'm doing wrong to prepare it? Thanks for any help
I have never worked used UCP-1 antiboby, but I can try to help you by asking you questions Do you know whether UCP-1 has different isoforms? Is it possible the 26kda is the short isoform of UCP1. Is the 26kda band appearing to all samples? Since its coming in your negative control, I suspect thats a non specific band and in that case just ignore it. perhaps those samples without 32kda is telling you that they don't express UCP1. Hope that helps
First of all I am going to tell you most commercially available antibodies will give you some non-specific bands. This is especially true for rabbit and goat antibodies. Another thing is the animal you are using. The proteins may have similar numbers of aminoacid but run differently in gel. I know my answer may not help you but hope you understand the antibody usually is not so specific as you expected.
Hi Jessica,
http://www.abcam.com/ucp1-antibody-ab10983.html
I looked at the datasheet for the UCP antibody that you are using and it states that weak bands could be detected depending on the BAT preparation. I would have suggested that the 26kda band you are seeing was a proteolytic cleavage product but since you see it in your negative control, this may be ruled out. What is your negative control? I would go back and look at your BAT prep and compare the preparation/storage method for the samples that do and do not contain the lower band.
Hope this helps. I know processing BAT samples are a bit tricky.
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