I am staining for neuronal microtubules in C. elegans strains expressing GFP-tagged beta-tubulin, using anti-GFP antibody (12A6, supernatant- DSHB). I observe no staining in any of my samples across multiple trials. The secondary antibody is either Alexa 488/564- both of which work well for other worm strains. My antibody dilutions (both primary and secondary) are either 1:50 or 1:100. I have tried different fixation methods: 2% PFA, Bouin's fixative, Methanol freeze-crack. I have also tried blocking with 0.5% & 1% BSA in PBST, and 5% & 10% NGS in PBST. None of these methods show any staining. I have also varied incubation duration for primary and secondary antibodies, with no results. The anti-GFP antibody works well with soluble GFP in C. elegans neurons, which leads me to believe that the antibody is unable to recognize/bind the epitope when GFP is tagged to another protein (either N-terminal or C-terminal).
Hi,
Your theory might just be correct. See here:
https://pdfs.semanticscholar.org/bc31/eb5ae94baf9480ef487c1c5892326d57f835.pdf
Have you seen these: http://www.wormbook.org/chapters/www_immunohistochemistry/immunohistochemistry.html#sec1_1
https://www.wormatlas.org/
Also:
https://www.cshlpress.com/pdf/sample/2013/Antibodies2/AB2FM.pdf (if you can find the book)
I hope this helps :)
Hi,
The antibody you're using has been produced using the native form of the GFP. See here: Article Generating a Battery of Monoclonal Antibodies Against Native...
In this article, the clone 12A6 was able to recognize a fusion-GFP protein (E-cadherin-GFP), but I guess this can't be generalize to all fusion proteins. I'm not convinced that your problem is due to the masking of the antigen by your fixative, but rather that the epitope recognized by this antobody is not present anymore in your particular system. I would advise you to try an other antibody before engaging in a tedious series of tests with antigen retrieval protocols. Try asking for test aliquots around you or directly from companies, as some of them provide them for free.
Maybe a polyclonal antibody would work better, since multiple epitopes can be recognized...In our hands, AB3080 from Millipore works well at 1/500 (it's a rabbit polyclonal antibody) in mouse brain tissue.
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