Unpredictable or absent signal despite following protocol. Results are inconsistent and do not match expected standard curves. Signal may vary dramatically between experiments.
Common Causes
1Mixing antibodies, standards, or detection reagents from different kit lots with different optimization
2Buffer formulations vary between kits and may be incompatible with mixed components
3Detection substrates from one kit may not be optimized for enzyme conjugates from another
4Sample type compatibility verified for one kit may not apply when mixing components
Solutions
1Use all components from the same kit and lot number for each experiment
2If components must be mixed, perform thorough optimization and validation with positive controls
3Keep complete kits dedicated to specific experiments; do not share components between projects
4Document all component sources and lot numbers to troubleshoot unexpected results