I am working with a dopamine transporter antibody for western blotting and it gives two bands: 50kDa for the reduced form and 80kDa for the non-reduced form. I was wondering if anyone could shed light on what these distinctions refer to?
Dear Kate,
According to one group's study (see link below), DAT has two states. The first is a mature, glycosylated form at ~85kDa that exists in the plasma membrane. The second is an immature, non-glycoslyated form at ~57 kDa that is found in the cytosol (note they used FLAG tagged DAT so untagged protein would be closer to to the 80, 50 split seen in the antibody spec sheet). If you take the mature form and remove all the N-linked sugars with a glycosidase, the protein goes back down to 57 kDa (see Fig. 1 and discussion in linked paper).
I think this is probably what you see with this antibody. However, to be sure what they mean by reduced versus non-reduced (as opposed to the mature versus immature found in this publication), you may reach out to the company and ask them for a response. If you get an answer, please let us know! Good luck!
http://www.pnas.org/content/98/18/10055.long
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