Distinguish small molecules from carrier proteins
Check buffers, stabilizers and carrier proteins in the formulation. Tris and glycine contain primary amines and can be removed by buffer exchange appropriate to target size. BSA and gelatin can themselves be labeled; use carrier-free material or a method that separates the target protein.
Calculate input after buffer exchange
Re-measure concentration and recovered volume after buffer exchange, check for precipitation or aggregation, then calculate dye input. Retain an unlabeled sample processed through the same exchange to distinguish preparation-related damage from labeling effects.