Lowry Assay for Assessing Protein Levels

Lowry Assay

A biochemical assay for determining the total level of protein in a solution. The method is based on the reaction of protein molecules with copper under alkaline conditions, followed by the reduction of the Folin-Ciocalteu reagent, leading to a colorimetric change that is directly proportional to the protein concentration.

Workflows Requiring the Lowry Assay:

Protein Extraction and Purification: In processes involving the extraction and purification of proteins from biological samples, the Lowry Assay is instrumental in determining the yield and purity of the protein at various stages.
Cell Culture: For cell culture applications, especially in assessing cell lysis efficiency and the protein content of lysates, the Lowry Assay provides essential quantitative information.
Biochemical Assays: In enzyme kinetics and other biochemical assays, the determination of protein concentration is crucial for calculating specific activity and for normalizing enzyme activity across different samples.

How to Perform a Lowry Assay

1. Preparation of Reagents

Alkaline Copper Solution: Prepare by mixing sodium hydroxide (NaOH), copper sulfate (CuSO₄), and sodium potassium tartrate in a specific ratio. This solution stabilizes the copper ions in solution.
Folin-Ciocalteu Reagent: Dilute with distilled water according to the manufacturer’s instructions. This reagent is sensitive to light and should be prepared fresh or stored properly.
Protein Standards: Prepare a series of protein standards (e.g., bovine serum albumin, BSA) in known concentrations to generate a standard curve.

2. Sample and Standard Preparation

Dilute your protein samples and standards to fall within the expected range of the assay sensitivity. Use a suitable buffer that does not interfere with the assay.
Pipette equal volumes of standards and samples into separate test tubes or wells of a microplate if using a micro-assay format.

3. Addition of Alkaline Copper Solution

Add a specific volume of the alkaline copper solution to each sample and standard. Mix thoroughly and incubate at room temperature. The duration of incubation may vary but is typically around 10 minutes. This step allows proteins to bind to copper ions.

4. Addition of Folin-Ciocalteu Reagent

After the incubation with the alkaline copper solution, add the diluted Folin-Ciocalteu reagent to each tube or well. This step must be performed with care, as the timing between adding this reagent to the first and last sample should be consistent across experiments.
Mix well immediately after addition and incubate at room temperature. The incubation period can range from 30 minutes to an hour, depending on the protocol. During this time, the reduction of the Folin-Ciocalteu reagent results in a color change.

5. Measurement of Absorbance

After the final incubation, measure the absorbance of each sample and standard using a spectrophotometer or plate reader set to a wavelength of 650 nm to 750 nm, typically around 660 nm.
Ensure the instrument is zeroed with a blank containing all reagents except the protein.

6. Calculation of Protein Concentration

Generate a standard curve by plotting the absorbance of each protein standard against its concentration. The relationship should be linear within the assay’s dynamic range.
Use the standard curve to determine the concentration of protein in your samples based on their absorbance values.

Alternatives to Lowry Assay

The Bradford and Bicinchoninic Acid (BCA) assays are two modern alternatives to the Lowry assay, offering simpler protocols and greater compatibility with various substances. The Bradford assay is favored for its rapid, one-step process, ideal for higher protein concentrations but sensitive to detergent presence. In contrast, the BCA assay, while slightly more complex, is more tolerant of detergents and reducing agents, making it suitable for a broader range of protein concentrations.