Check the reaction system
Confirm a terminal alkyne on the target and an azide on the dye. Review copper, ligand and reductant additions and preparation times. Chelators or other copper-binding buffer components can affect catalysis. Correct omissions, formulation errors or mismatched handles before comparing dye input.
Measure conversion and sample loss
Assess conversion with an analytical method that resolves conjugate from free dye, while checking precipitation, recovery and function. Higher total fluorescence may simply reflect more free dye. Change one condition at a time and compare purified products.
Separate conversion from sample damage
Keep target concentration and the analytical method fixed and change one factor per comparison. Assess compatibility of the copper catalyst system with the sample separately.
| Observation | Check and action |
|---|---|
| No detectable product | Confirm terminal-alkyne/azide pairing and catalyst components; azide-only or DBCO reactions require a different pairing or workflow |
| Low conversion, intact sample | Check catalyst preparation, chelator carryover and reactant concentrations |
| Product forms but activity falls | Use a catalyst-treated control without dye to assess oxidation, precipitation and residual copper |
| High total fluorescence, weak product peak | Remove free dye and assess conversion using an assay that resolves substrate and product |