The Lowry protein assay is a common method used to measure the protein concentration in products. In this lesson we will learn the steps in this method and how the reaction works.
Lowry Protein Assay Principle
When researchers investigate the effects of diseases or the health of an individual, the protein concentration of a patient’s blood serum is often tested. Several different methods can be used to test the protein concentration. One of these is the Lowry protein assay, one of the most common methods used to measure the concentration of protein in a sample.The Lowry protein assay uses copper, which bonds with the peptide bonds in proteins under alkaline conditions. This forms a monovalent copper ion which can then react with the Folin reagent, which in turn can be reduced into a blue colored substance.
This blue color can be measured using a spectrophotometer to determine the concentration of blue in the sample. Thus, the concentration of protein can be determined.
Steps in Reaction
Let’s go over the steps in the reaction. First a standard needs to be prepared. Oftentimes bovine serum albumin (BSA) is used as the standard. The standard and sample follow the sample steps for measuring the protein content. Since the protein content of the BSA is known, it can be used to convert the absorbance obtained from the sample to protein percentage in the sample.
Then the Lowry solution needs to be made. It consists of an alkaline solution, copper sulfate, and sodium tartrate. Then the Lowry solution can be mixed with the sample.
This mixture needs to be incubated for 20 minutes to ensure that the reaction occurs. At this point the reaction is yellow. The Folins reagent is then added and, once again, the mixture is incubated.When this sample is removed from the second incubation we will be able to see a blue color if there were proteins present in the sample. The concentration of blue color can then be measured using a spectrophotometer.
A wavelength of 750 nm will typically be used, but if high concentrations are expected, then 500 nm can be used. High concentrations won’t work with a 750 nm wavelength because the absorption will be beyond the ability of the machine to read it. The lower wavelength is better at measuring high concentrations because it measures a wavelength of light that appears in a lesser amount in the reaction.
Whichever wavelength is used, the same one needs to be used for both the standard and the sample.
Mechanism of Reaction
The copper in the Lowry solution is able to react with the peptide bonds in the sample to form a peptide-copper bond. When this bond reacts with the hydroxide ions in the Folins reagent, the copper is reduced to copper (II). The copper (II)-peptide compound has a blue color.Copper is able to bind to the four peptide bonds because it has extra electrons and the nitrogen-hydrogen (NH) on the peptide bond has an empty orbital.
The copper is able to be reduced by the Folins reagent because copper prefers to be in the 2+ state, so electrons can easily be removed by even a weak base. The exact mechanism by which the Folins reagent reduces the copper is not known.
The Lowry protein assay is one of the most common methods used to measure the concentration of protein in a sample. It occurs because the copper in the Lowry reagent can react with peptide bonds. These bonds become visible when the copper is reduced, because this forms a blue colored solution.
The concentration of blue color can be measured with a spectrophotometer. The steps in the Lowry protein assay are:
- Prepare Lowry solution
- Mix Lowry solution with sample or standard
- Incubate for 20 minutes
- Add Folins reagent
- Measure absorbance at 750 nm, or 500 nm for higher concentrations