We have started to get some issues that we didn’t have before and its getting quite annoying so wanted to check if someone have experience with this before.
HeLa cells where we have previously used AZdye594-DBCO to label azides attached to proteins in the nucleus. These are fixed and permeabilized cells. For a couple of weeks we have gotten this strong background and absolutely no labeling in the nucleus. As if the dye gets soaked up and dont even reach its target. The signal is very strong, not dim at all as one would have from normal stickiness of fluorophores to proteins. We get the same background binding in negative controls as well where there is no azide present to bind to.
I suspect thiol-reactivity due to the DBCO group. But it could also be something else.
Any advice or experience with issues like this before? We have already excluded that the fluorophore could have gone bad by opening a fresh batch.
Any advice or pointers much appreciated! Posting chemical structure and LC-MS in responses to this thread as a new user I am not allowed to post multiple images in a single post.
