| | Re: non specific binding of FITC to erythrocytes
I am experiencing unspecific binding of my FITC to my streptavidin-coated microspheres too. Basically, I have streptavidin-coated microspheres to which biotinylated DNA and biotinylated anti-HA antibodies bind to. The biotinylated DNA actually has a sequence of HA at the 3' end, so after in vitro translation, peptides with HA tags are produced and they bind to the anti-HA antibodies on the beads. I detect these proteins using a peptide that is conjugated to FITC. During my analysis with flow cytometry, I realized that I get varying signals.
And in my negative control, there is supposed to be no DNA at all so no proteins are produced and no peptide-FITC should bind. But I get fluorescence signals in the negative controls and this indicates unspecific binding.
Been stumped at finding a way to get rid of the unspecific binding, though the only thing for now I can think of is to perform more stringent washings in between each step.