Primary-amine labeling
NHS esters react with accessible primary amines on lysine side chains and protein N-termini. Tris and glycine compete for dye; BSA and gelatin can also be labeled. Remove small molecules by suitable buffer exchange. Carrier proteins require separate purification or a carrier-free sample.
Free-thiol labeling
Maleimide requires accessible free thiols; cysteines in disulfide bonds do not react directly. If reduction is needed, check integrity, aggregation and function afterward, and address reductant carryover. Multiple available thiols can produce multiple labeling sites.