Confirm free thiols
Check disulfide bonds, thiol protection and accessibility. Before a thiol assay, check whether buffer reductants interfere with the readout. Total cysteine count in the sequence is not the available thiol count.
Separate reduction damage from labeling effects
Compare untreated, reduction-only and reduced-and-labeled samples for recovery, aggregation, and binding or enzyme activity. If function already declines in the reduction-only sample, address pretreatment before increasing dye input.