We are using an alexa 488 probe to mark a membrane protein and we want to see whether we can induce endocytosis. Since endocytosis in this case is not very obvious we would like to kill the extracellular signal to see whether the endocytosed molecules are protected, so we could quantify how much was internalized.
From the Thermo-Fisher web site:
What can quench Alexa Fluor® dyes?
Just like all dyes, Alexa Fluor® dyes are susceptible to typical quenchers such as chromogens (trypan blue), quenchers (QSY®), and other compounds such as 1, 4-diazabicyclooctane (DABCO) and N-propyl gallate (NPG), which, ironically, are also used as antifades.
The question, then is whether any of these quenchers is compatible with your purpose.
Almost all fluorescent probes are subject to photobleaching (especially a problem at high lens magnifications).
I would try imaging the cells for a short period with a 10x or 20x lens under 488 nm excitation i.e. 5 - 10 minutes. Then have a look and see if this has gone down. If you have a mercury lamp as a white light source I would try this rather than a laser as it may reduce the stress (frying) on the cells.
Not sure if this will suite your purposes but I always have problems with loss of signal so this approach may work when you want to lose signal!
However I suspect that the conditions for photobleaching will also kill the signal in the endosomes etc so not sure how much this will help.
Ideally you would probably need a fluorescent dye conjugated to your antibody that changes fluoresence or fluorescent signal at a different/lower pH. That way the "background" signal in the plasma membrane won't be such a problem. Not sure if this is worth looking into (?).
Although you might already be doing this having control cells that have been treated with endocytosis inhibitors may show up some interesting differences.
Sorry I can't be more help!
Gary
Adam, thanks for the help. I had found that info, but all those look pretty harsh for the cells, so I don't think I can use them, don't you agree? At least not for live imaging.
Gary, I agree in that it is very likely that I will kill the vesicles signal too. The second option is interestings. Something like phluorin. In our case this is a toxin already bound with alexa, so no antibodies present. Still I think it is worth thinking about it for a modification to our toxin.
The third idea is perfect. Something like dynasore. I don't have it right now, but I will buy it right away.
Thanks both of you for the suggestions.
Cheers
How about an anti-Alexa antibody?
https://www.thermofisher.com/antibody/product/Alexa-Fluor-488-Antibody-Polyclonal/A-11094
Well, that's a good idea! Sometimes you just try to look for the most sophisticated and complicated possibilities...Thanks Adam!
Hi Marcelo,
I would also suggest the anti-Alexa488 antibody mentioned in a previous post. Alternatively, quenching by potassium iodide is a very cheap alternative as shown by the attached Stern-Volmer plot we measured. Although I don't know the membrane permeability of iodide.
Peter
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