A researcher is attempting to determine the concentration of a protein in an unknown sample using the Folin-Ciocalteu method. They prepare a standard curve using known concentrations of a standard protein solution and measure the corresponding absorbance values. What is the most critical factor the researcher must ensure when generating the standard curve for accurate quantification of the unknown sample?

The researcher must make sure that the standard protein solutions are prepared and measured under exactly the same conditions as the unknown sample—same buffer, same reagent lot, same incubation time, temperature, and path length. This guarantees that the absorbance values for the standards and the unknown are directly comparable. If the conditions differ, the absorbance‑concentration relationship will shift and the curve will no longer match the unknown. For example, if the standards are incubated for 30 minutes while the unknown is only 20 minutes, the resulting curve will over‑estimate the unknown’s concentration. Thus, identical experimental conditions for both standards and sample are essential for accurate quantification.