What is the correct sequence of steps for measuring protein concentration using an alkaline copper solution in a colorimetric assay?

First, prepare a set of protein standards of known concentration and your unknown samples, then add the alkaline copper solution to each tube. Next, incubate the tubes for the recommended time (usually 10–15 minutes) so the copper ions can react with the protein’s phenolic groups, forming a blue complex. After this incubation, add the Folin‑Ciocalteu reagent, which reacts with the copper–protein complex and intensifies the blue color. Finally, measure the absorbance at 595 nm, plot the absorbance of the standards to generate a standard curve, and use that curve to calculate the protein concentration in your samples. For example, if a 1 mg/mL standard gives an absorbance of 0.