Lowry Method: A Protein Assay

Although Lowry method is not good enough compared to Bradford and Bicinchoninic Acid method for protein quantitation, but it is still widely used due to its acceptability to estimate protein in almost all circumstances in which protein mixtures or crude extracts are involved. Lowry method is based on the conversion of Cu²⁺ to Cu⁺ under alkaline conditions. The reactions result in a strong blue color, which depends partly on the tyrosine and tryptophan content. Sensitivity of the method is down to about 0.01 mg of protein /mL, and is best used on solutions with concentrations in the range 0.01-1.0 mg/mL of protein. If your sample contains protein less than 0.01, then you can used Bradford method (it can be used for measuring between 1 and 10 micrograms of protein, for microassay) and Bicinchoninic Acid method (it can be used for measuring between 0.5 and 10 micrograms of protein/mL, for microassay).

Material

1. Complex-forming reagent: Prepare immediately before use by mixing the following stock solutions in the proportion 100:1:1 (by vol), respectively:
   Solution A: 2% (w/v) Na2CO3 in distilled water.
   Solution B: 1% (w/v) CuSO4•5H2O in distilled water.
   Solution C: 2% (w/v) sodium potassium tartrate in distilled water.
2. 2 N NaOH.
3. Folin reagent (commercially available): Use at 1 N concentration.
4. Standards: Use a stock solution of standard protein (e.g., bovine serum albumin fraction V) containing 2 mg/mL protein in distilled water, stored frozen at –20°C. Prepare standards by diluting the stock solution with distilled water. You can make the solutions with several series concentration, for example 0; 10; 20; 50; 100; 200; 500; 1000; 2000 micrograms/mL.

The Method are:

1. To 0.1 mL of sample or standard, add 0.1 mL of 2 N NaOH. Hydrolyze at 100°C for 10 min in a heating block or boiling water bath.
2. Cool the hydrolysate to room temperature and add 1 mL of freshly mixed complex-forming reagent. Let the solution stand at room temperature for 10 min.
   • The reaction is very pH dependent, and it is therefore important to maintain the pH between 10 and 10.5. Therefore, take care when analyzing samples that are in strong buffer outside this range.
   • The incubation period is not critical and can vary from 10 min to several hours without
     affecting the final absorbance.
3. Add 0.1 mL of Folin reagent, using a vortex mixer, and let the mixture stand at room temperature for 30–60 min (do not exceed 60 min).

   The vortex-mixing step is critical for obtaining reproducible results. The Folin reagent is reactive only for a short time under these alkaline conditions, being unstable in alkali, and great care should therefore be taken to ensure thorough mixing.

4. Read the absorbance at 750 nm if the protein concentration was below 500 micrograms/mL or at
   550 nm if the protein concentration was between 100 and 2000 micrograms/mL.
5. Plot a standard curve of absorbance as a function of initial protein concentration and use it to determine the unknown protein concentrations.
   • The assay is not linear at higher concentrations. Ensure that you are analyzing your sample on the linear portion of the calibration curve.
   • A set of standards is needed with each group of assays, preferably in duplicate. Duplicate or triplicate unknowns are recommended.

One disadvantage of the Lowry method is the fact that a range of substances interferes with this assay, including buffers, drugs, nucleic acids, and sugars. In many cases, the effects of these agents can be minimized by diluting them out, assuming that the protein concentration is sufficiently high to still be detected after dilution.

You can also increase Lowry method’s sensitivity by doing these two following things:

1. If the Folin reagent is added in two portions, vortex-mixing between each addition, a 20% increase in sensitivity is achieved.
2. The addition of dithiothreitol 3 min after the addition of the Folin reagent increases the sensitivity by 50%.

Regards.