How to Apply the Lowry Method of Cell Concentration

The Lowry method is a biochemical assay used to determine total protein concentration. The test is a colorimetric test meaning that the results are interpreted based on the intensity of color in the test solution. It works through the reduction of Cu²+ ions to Cu+ ions by the peptide bonds of proteins and produces a violet color under alkaline conditions (pH greater than 7). In addition, Folin reagent, which is composed of inorganic salts, is added and reacts with residues of the amino acids tryptophan and tyrosine producing a blue-green color. The amount of protein is directly proportional to the intensity of the color observed from these two reactions. The combination of these two reactions together produces a very sensitive test that can give accurate results down to 0.01 mg/mL of protein. There are several steps that must be taken to apply the Lowry method correctly.

Instructions

    • 1

      Place at least three different known amounts of protein into three respective measured volumes of water along with a fourth sample that contains only water. The volume should be the same in the three different solutions containing known amounts of protein and the control sample without protein. The amounts used will vary between experimental procedures. These will be used as the standard curve of comparison in calculating protein concentration.

    • 2

      Add a specified amount of alkaline copper reagent to each solution and mix well. This product contains the Cu2+ ions that oxidize the peptide bonds of the proteins producing a violet color. The amount used will vary again based on the preference of the volume of protein solution from Step 1.

    • 3

      Wait a minimum of 10 minutes to allow the reactions to proceed to completion.

    • 4

      Add a specified amount of Folin reagent to each solution, mix thoroughly, and allow each to sit for 30 minutes to an hour to let the reaction run to completion. Again, amounts will vary based on the specific experiment.

    • 5

      Use a spectrophotometer to obtain the absorbance value for each sample. The photometer is normally used in the range of 500nm to 750nm for this type of assay. Spectrophotometer information is located in the Resources section.

    • 6

      Plot the resulting absorbance values versus the known protein amounts for the respective solutions to determine the protein concentration.

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