I am using an Abcam (ab20066) anti-D1 antibody as well as a Millipore (AB5084P) anti-D2 antibody for Western blotting of PFC and striatum membrane fraction homogenates. Unfortunately, there is a lot of non-specific binding. Particularly frustrating is a doublet right around the 50 kD mark in the D1 ab (which is the region of interest). The Abcam rep suggested that the lower band is probably D1 while the upper band might represent a post-translational modification (acetylation was mentioned). This idea really intrigues me and I'd like to explore it further. My idea was to run a gel with purified receptor and acetylated receptor and compare the migration rate to what I'm seeing in my homogenates. Problem is, I can't seem to locate any purified D1 receptor (although, I have found a purveyor of recombinant D2r).
Does anyone have any other ideas as to how I could go about characterizing my bands and/or post-translational modifications of my receptors of interest?
Further, does anyone know anything about dopamine receptor acetylation (i.e. function). My PubMed searches have been fruitless thus far.
Many thanks!
We (and many other labs) can give you overexpressing D1 or D2 cell lines that you can compare with WT lines if that would help. We have never studied acetylation or heard of it for DARs, but problems with DAR antibodies are not unusual.
Are you sure that the receptors are not phosphorylated. Both D1 and D2 can be phosphorylated and this seems more likely than acetylation.
I struggled for a long time to get good Western blot data using D1R Abs on brain extracts. For some recent findings on D1R blotting using membranes from brain, look at Molecular and Cellular Neuroscience 46 (2011) 645–654; I'd be happy to provide more information.
I have thought about both phosphorylation and glycosylation...both of which would add an interesting spin to our findings. I'll check out the reference from Molec. and Cell. Neuroscience. Thank you!
I am using mouse monoclonal antibody for D1 and polyclonal antibody for D2. And i got very good and clean bands in mouse brain tissue homogenate. you can see the bands in the attach file.. It will be very grate full if anyone share your protocol for isolating cytosolic and membrane fraction from brain tissue..
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