Did sample handling erase the protein signal?

Both split aliquots should undergo the same denaturation protocol, with only the delay before denaturation differing. Higher signal after immediate denaturation then supports loss during that delay; otherwise, denaturation timing and processing chemistry are confounded.

Use two aliquots from the same homogenized sample, denaturing one immediately at collection and processing the other after the usual delay, then assay them together within the validated range. A higher signal in the aliquot denatured immediately at collection supports handling-dependent loss; similarly low signals leave low biological abundance plausible. The uncertainty that could flip that interpretation is whether delay was truly the only difference, since unequal starting material, storage, dilution, or assay conditions would break the paired comparison.

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.

Higher signal in the immediately denatured aliquot than in the delayed aliquot supports handling-dependent loss, provided both came from the same starting sample and were assayed together. Did those two controls differ only in processing delay?

Higher signal after immediate denaturation supports handling loss, so the practical response is to revise sample processing rather than interpret the stored lysate’s weak signal as low abundance.

Higher signal in the immediately denatured aliquot than in the delayed aliquot supports handling-dependent loss, provided both began from the same sample and were assayed together.