Hi ,
I have to transfect cells with a GFP tagged plasmid and want to look under the microscope to see the localization of the protein in both transfected and non-transfected cells. How can I do that? should I use antibody against my protein in nontransfected cells? because my antibody is not fluorescent . please help :(
I would say this is a very basic question that you should discuss with your teacher or advisor, as it sounds like you're unclear about lab work that you've been assigned. In short, the standard method for tagging specific proteins (other than the fluorescent proteins already transfected into a living organism) for fluorescence microscopy is immunostaining, and indeed, you'd use antibodies - probably primary *and* secondary antibodies, with the secondary antibodies having an attached fluorophore. (There are primary-only protocols, but that entails much less signal, so it's rarely used.)
But I don't know about your specific experiment. Are you sure you're not just looking expression vs non-expression of GFP? In that case, the non-transfected cells require no special treatment, and you'll probably look at the cells under both phase contrast or DIC and then fluorescence.
Have a look at a good intro to immunostaining - there are many out there in textbooks or on the web - here's one example:
https://www.thermofisher.com/ie/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/ihc-immunodetection.html
Peter Werner thank you so much for your answer! You actually cleared up some of the confusion I had. I am over expressing my protein in order to see if there is any difference in its localization compared to the wild type. that is why I am wondering if I should use antibodies for it in my wild type since there is no GFP to spot it
Hi, If you already trasfected the cells by plamid containing GFP, just look at the cells under fluorescent microscope.Thats all. You dont need immunostainig anymore.
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