Remove thiol-containing reductants
DTT and β-mercaptoethanol compete with target thiols for dye. Use suitable desalting or buffer exchange before conjugation, then recheck recovery and free thiols. Record the interval before dye addition to help identify reoxidation.
Check residual TCEP
TCEP contains no thiol but can react with maleimide. Removal requirements depend on residual concentration and the validated procedure. If compatibility data are unavailable, compare labeling with and without TCEP carryover rather than assuming compatibility.
Document reductant removal
Record starting reductant concentration, recovered volume, exchange method and delay before dye addition. Assess DTT, β-mercaptoethanol and TCEP separately; a desalting step does not by itself demonstrate complete TCEP removal.