I have ground up rat plantar skin for western blots. I wish to probe for a neurotrophin. They look terrible, lots of spurious bands at the concentration needed to detect my protein of interest. I think that maybe there is so much collagen, matrix etc in the samples that antibodies stick like crazy. Does anyone have any thoughts?
Hi Samantha,
Could you tell us in detail the extraction protocol, how much protein you are loading, the antibody dilutions etc and show an image of one of the blots, it will help everyone to troubleshoot your western.
Cheers
Jacob
Sorry, i thought my description of lots of spurious bands was enough, I will add a photo.thanks.
Even without the protocols you may well be right. The vast majority of protein in that extract will be epidermal keratins and dermal collagens, with living cells a very minor component. So a rare molecule like a neurotrophin may well be a major challenge to detect. Not surprising if you get high background when you would have to push up the antibody levels and exposure time to high levels. Why not try IHC instead? The signal might then be localized to a very small region (if present), but still bright.
I haven't worked with skin homogenate, but with cultured cell lysates I've definitely seen BSA showing up when it shouldn't. There's just so much of it, and it's sticky.
Have you tried optimizing the blocking buffer? That may help reduce the spurious bands and improve detection of your target. It's incredible what a huge difference the blocking reagent can make in background banding.
probably epidermal keratin and desmoglin/ desmocollin interfere in the process.
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