Identify competing biotin
Review small-molecule removal after biotinylation and biotin in culture media, storage buffers or additives. Free biotin can occupy binding sites during detection; simply adding more fluorescent streptavidin may not solve the problem.
Account for sample loss
Remove free biotin using a separation method appropriate to target size, then re-measure target concentration. Compare signals at matched target input so purification losses are not mistaken for altered binding.
Distinguish target-bound from free biotin
Retain biotin attached to the target while removing excess free biotinylation reagent and small-molecule biotin. Assess target recovery after cleanup. Buffer exchange is interpretable only when the chosen method effectively separates small molecules from the target.