[Drosophila] anti-GFP on polytenes
(by imcdanie from ScrippsCollege.edu)
Mon Oct 8 16:31:03 EST 2007
I am currently using the AbCam 290 anti-GFP antibody to look at a GFP-tagged protein on polytene chromosomes. I see excellent signal with very good specificity on most sets of chromosomes on the slide. However, I occasionally find a set of chromosomes with ZERO GFP-signal, not a single band. These can be fully extended sets, so it doesn't appear to be correlated to unbroken nuclei. In some cases, this set is touching or surrounded by other sets that have a very bright signal.
I see this regardless of the concentration of anti-GFP Ab that I use. I also see this highly specific signal-loss when I do not use antibody at all, and simply look at the GFP signal alone. These chromosomes have been fixed in formaldehyde (~2% for 5 minutes, in 45% acetic acid). When I saw the isolated signal disappearance on slides that had not been treated with antibody, I assumed it was caused by fixation or drying out, and one set of chromosomes was affected more than the others. However, I didn't think fixation or drying out would affect the epitope recognized by the antibody.
Has anyone ever heard of any problems like this when working with GFP? Or is it more likely to be an issue with the expression of my tagged protein?
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