With the ReadyProbes Avidin/Biotin Blocking Solution (1X), why is there a step to add biotin to the sample?
The first step is to bind all endogenous biotinylated entities with an unlabeled avidin. The free D-biotin is then added to bind to all biotin binding sites on this added avidin. The free biotin will be washed away before any other labels are added and all biotin binding sites will be occupied on the unlabeled avidin.
I am labeling cells with a biotinylated primary antibody, followed by an avidin dye conjugate, but I'm getting non-specific mitochondrial labeling, even with an avidin-only control. Why is this?
Mitochondria have endogenous biotin, which is being labeled by the streptavidin. You will need to block the endogenous biotin first, after you fix and permeabilize, but before you block for your primary antibody.
How is ReadyProbes Avidin/Biotin Blocking Solution (1X) different from other endogenous biotin blocking kits?
The only difference between the ReadyProbes Avidin/Biotin Blocking Solution (1X) and other endogenous biotin blocking kits is the ease of use. In the ReadyProbes Avidin/Biotin Blocking Solution (1X), we provide the two components already pre-mixed and ready to use.