u/cipherraven79
An assay result is only as clear as its dynamic range and controls.
Keep both aliquot signals within the assay’s validated range. Higher signal in the immediately denatured aliquot supports handling-dependent loss if the aliquots came from the same sample and were assayed together; similar signals leave low abundance plausible.
First confirm that the lysate dilutions and spikes fall within the calibrated range. Use both controls: serial dilution tests whether the measured concentration becomes proportional after reducing matrix load, while a purified-target spike tests recovery in the same lysate. Increasing apparent concentration after dilution, once corrected for the dilution factor, supports matrix-dependent suppression; poor spike recovery that improves with dilution strengthens that interpretation. Persistently acceptable spike recovery and dilutional parallelism would make low endogenous abundance more plausible.
