Good morning,
we are performing an intracellular click-reaction using an azide coupeled to an intracellular protein and a TAMRA alkyne (after cultivation, the cells are fixed, permeabilized, blocked with 3% BSA, and incubated with the click-reaction cocktail - with lots of washing between the steps). We get a good signal, however, we have massive unspecific binding of the dye when we add it to cells which do not exhibit the azide. We thought that it may be due to the dye and changed it to AF647, but we got the same result.
When we washed the cells with high amounts of DMSO (up to 50% v/v), we were able to wash some of the unspecifically bound dye away, however, it's still not perfect.
Does any of you have an idea how to supress unspecific binding? It would be very much appreciated!
Regards
Leonhard
So in the fixed, but unlabelled cells the dyes are binding? What was the fixation?
Yes, i forgot it. I labelled nuclei. It worked fine but it was an azide stain. [Now i just read that methanol can be used as fixative, too (on the website as protocol tip).]
Do the labelled and unlabelled cells have the same "background"? If not, might be the reaction (or a side-reaction rather) just searching desperately some partner to act with.
Good afternoon Leonhard,
did you solved this problem? I have a same question. For several weeks I try to solve this, but unsuccessfully.
I'll be glad for any advice, if you know something more.
Hey, I am having the same problem of AFdye-azide non-specifically binds cellular proteins. Planning to wash the cells with Urea or Guanidine hydrochloride (disrupt hydrophobic interactions), post labeling. Haven't done so yet. Have you tried Urea wash(i.e., 5%)?
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