I labeled an antibody with FITC a year ago, titrated it for flow and it gave good results. I aliquoted it and store it at -20C away from light, as the kit recommends. I also have it wrapped in foil. I recently took an aliquot for an experiment and re-titrated it but this time it looks as if the fluorescence was so dim for all the concentrations it was almost nonexistent.
I am wondering if I should just re-label the antibody, but that would put the antibody through a freeze-thaw cycle. What should I do...relabel anyway? Should I instead use a secondary FITC conjugated antibody from now on?
Hi Justine,
During storage at -20 your labeled antibody can deteriorate anyway. Did you check if the antibody is still active? Since the fluorescence of fluorescein is pH dependent you could also check the fluorescence in buffers with a pH>7.4. Furthermore, relabeling in general does not work since you already have covalently bound your dye to the antibody previously and these amines are hence not available for labeling anymore. Of course it depends how many dyes you have on average on your antibody. If you have only two, you might give it a shot, but I would buy new fresh antibody.
Best regards.
Hi Justine,
I would buy secondary antibody and use that. They are cheap, stable and you get a lot for your money. Plus you can choose from a number of different fluorophores besides FITC. I avoid using FITC if I can because it is dimmer compared to PE or APC which I prefer for flow.
Good Luck!
Thanks, I will try a secondary antibody then to use then. It has to be FITC though because it is part of a multicolor experiment.
Hi Justine,
Usually,the antibody should not be stored at -20 degree if it is conjugated with any fluorescence dye. Besides, you should use antoclaved reagents/buffers for the fluorescence conjugation. You could choose AF488 instead of FITC. And Based on my experience, re-labeling is useless.
Wish your project goes well.
If you are looking for accuracy, don't waste your time and money. Otherwise you could go for secondary antibodies which may or may not work as well. The light exposure is not the only factor that could affect the signal, also oxidation and prolonged freezing because some fluid continue to evaporate even at cold temperatures and affects the solution at the end.
I have no experience in flow cytometry, but I think usage of secondary antibodies is cheaper, easier and more flexible.
Just a suggestion to find out, if your FITC label or your labelled antibody is the problem: Try to detect the labelled primary antibody which does not give you any signals with a secondary antibody directed against the species. If you get a signal, you know that it's "only" the FITC label that is not stable, if you don't get any signal although you are sure that the antigen is there it's most likely that your antibody is bad.
Vivica, I did exactly that. I tested my antibody with a secondary antibody using flow. I got a signal, but it appears that the primary antibody is binding nonspecifically to other cells it is not supposed to bind to, so I think it's time for fresh antibody.
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