Determination of Protein Concentration of a Solution Lowry Assay Determination of Protein Concentration of a Solution

What is the Lowry Assay? Method created by Oliver H. Lowry in the 1940s Involves mixing a series of chemicals with a sample of a protein solution under particular conditions In the presence of proteins, a chemical reaction occurs that leads to the formation of a blue color This type of assay is referred to as a colorimetric assay The intensity of the blue color is proportional to the amount of protein present in the sample (i.e. the higher the protein concentration, the darker the blue color)

Lowry Assay Results High concentration of protein No protein

What materials are needed to perform a Lowry Assay? Reagent A (consisting of sodium carbonate and sodium hydroxide) Sodium potassium tartrate solution Cupric sulfate solution Test tubes or cuvettes Protein sample (unknown), diluted Set of protein standards (usually BSA—Bovine Serum Albumin) prepared in a buffer solution at varying concentrations (0.0mg/ml -2 mg/ml) Incubator or waterbath set at 37°C Spectrophotometer

How is Color Intensity Converted to Data? The absorbance value for each sample is determined using a spectrophotometer. The darker the blue color, the higher the absorbance value.

How is the Lowry Assay performed All reagents are prepared at the concentrations noted in the protocol. The protein standards are prepared by performing several dilutions of a stock standard solution (usually 2mg/ml) to create standards in a series of known concentrations. 2-3 dilutions of the protein sample to be tested (i.e. the “unknown” sample) are prepared. Measurements outside of the absorbance range of 0.05-2.0 are not very accurate. Because we don’t know what the protein concentration is, we perform several dilutions in the hope that one or more will fall within the acceptable range for the assay. The reagents are mixed with the protein standards and unknown samples in triplicate and incubated at 37°C for 30 minutes. For each sample, three test tubes are prepared that will give us three spectrophotometric measurements. We perform the test in triplicate to improve accuracy. If a mistake is made, it is more likely to be discovered when the test is performed in triplicate. The absorbance of each sample is measured by the spectrophotometer and the measurements are recorded.

Why do we need to measure standards? By measuring the absorbance of standards with a known protein concentration, we can create a standard curve. There is a linear relationship between the absorbance of the sample and the concentration of the protein in that sample. We can use the equation of the line generated by the standard curve to calculate the concentration of unknown samples, given the absorbance of those samples By using multiple standards, we improve the accuracy of our standard curve