Confirm target-bound azides
Review azide modification and purification records. Samples containing only amines or thiols need an azide handle for this route. If the target carries DBCO, use an azide dye instead.
Remove competing azide sources
Check for residual modification reagent and sodium azide in storage buffers. Remove them by purification or buffer exchange suited to target size, then re-measure target concentration before calculating input. More DBCO dye does not replace removal of competitors.
Distinguish sodium azide from target-bound azides
Organic azides attached to the target are intended coupling sites; sodium azide in storage buffer is a free competitor. Re-measure target concentration after small-molecule removal. Required exchange cycles depend on starting concentration, volume and separation method; do not transfer a fixed cycle count from another kit.