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How are fluorochromes like FITC conjugated to antibodies? Are they covalently bonded? If they are covalently bonded, will low temperature (-20 Celsius or lower) break the covalent bonds and detach the fluorochrome label from the protein?

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Fluorochromes are covalently bound to the antibodies to form stably labelled molecules. This paper describes it in great detail, from the abstract:

Fluorochromes can be covalently conjugated to antibodies through reactions with thiol or amine groups. Typically, fluorochromes containing isothiocyanate, succinimidyl ester, or sulfonyl chloride reactive groups are conjugated to amines on the antibody molecules.

These covalent bonds are stable at room temperature and are also at -20°C. It's more likely that repeated freeze-thaw cycles damage the antibody.

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  • $\begingroup$ most of the direct-conjugated antibodies for cytometric analysis I've purchased usually specifically point out "Do not freeze". I am recently staining FITC labeled peanut agglutinin for B220+ B cells. The labeled PNA was stored in 4C but when I took it out, it was in frozen form. I've tried dilutions to 1:1000 but there doesn't seem to have enough negative population compared to negative control. Almost all stained populations displayed fluorescent intensity over 10^2. I was worried if the freezing caused the fluorochrome to detach from the PNA protein. $\endgroup$ – eu4z0d Dec 7 '16 at 8:42
  • $\begingroup$ @AlexB I think it is more the repeated freeze-thaw cycles which are problematic for the antibody. The labelling is pretty stable as far as I know. $\endgroup$ – Chris Dec 7 '16 at 8:55
  • $\begingroup$ @AlexB is the FITC-PNA in DMSO or aqueous solution? If it's DMSO, it'll freeze at 4°. $\endgroup$ – MattDMo Dec 7 '16 at 15:36

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