Purification and labeling density
Remove free dye before measuring DOL with the appropriate extinction coefficient and absorbance correction. Record target recovery. DOL gives average labeling density; determining site distribution requires a separate analysis suited to the conjugate.
Stability in the intended medium
Maleimide–thiol adducts can undergo exchange in thiol-containing media. Compare fresh and stored conjugates for increased free dye, aggregation and loss of function, recording buffer, temperature and time. Establish storage limits from that conjugate’s results.