Choose purification for the target size
Desalting or size-exclusion separation may suit proteins much larger than the free dye. For short peptides and small products, verify that the method actually resolves both species. Also check whether residual CuAAC catalyst components affect downstream assays.
Locate background with controls
A matched sample without an alkyne, exposed to dye and purified identically, reveals adsorption or carryover. A no-dye sample measures baseline fluorescence. Compare all groups at matched target concentration, exposure or gain.