I have been doing literature research and haven't been able to determine what FITC alone stains in yeast cells. It may just be nothing, but from all information i have seen, FITC is only seen in imaging when used conjugated to something like ConA or Dextrin. I hope to determine some control for an upcoming experiment. Does anyone know?
Thanks for any help!
FITC alone is a fluorescent molecule. It's commonly used as the secondary antibody since its fluoresces at the 488 range allowing you to visualize whatever you've stained for with your primary antibody. On it's own, applying FITC shouldn't stain anything (unless you turn the gain way up and are accidentally visualizing autofluorescence of your tissue), it's merely used as a read-out of whatever you're targeting with it. So if you're using a Rabbit anti (protein of interest), you can use a FITC secondary antibody designed against rabbit (or whatever animal your primary was raised in). Additionally, you can custom design or order antibodies that have FITC fluorophores directly attached to them, and forgo the secondary application altogether.
Thanks Kristyn,
I can understand that, but let's take Rhodamine123 for example. That's a fluorescent molecule too, correct? I know when we use them with yeast cells, they stain the mitochondria with out pre-treatment to conjugate to antibodies. So, besides the colors they fluoresce at and the structure of the molecules, what separates these two? why would one stain an organelle when introduced and the other (FITC) not?
Good job on your dissertation defense the other week too btw.
Hm, I'm not sure. My vague understanding of Rhodamine is that it is mitochondria specific while FITC is not specific to any organelle, but rather is based on what you're staining for with the primary (at least as I understand it). Wish I could be of more help, and thanks!!
ok. Thanks, I thought FITC was non-specific from the literature, but now i feel better about it.
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