How to determine the amount of protein in a sample?

December 22, 2025 •

5 min read

The precise quantification of proteins is an essential step in countless research fields, from drug development to nutritional analysis. Understanding how to determine the amount of protein in a sample is therefore a fundamental skill for any scientist or analyst working in a laboratory setting.

Quick Summary

Several analytical techniques are used to quantify protein concentrations, from colorimetric assays like Bradford and BCA to nitrogen-based methods like Kjeldahl and Dumas. The ideal choice of method depends on factors such as sample composition, sensitivity requirements, and potential interfering substances in the mixture.

Key Points

Method Selection: The right quantification method depends on your sample's specific properties, desired sensitivity, and the presence of any interfering agents.
Colorimetric Assays: Bradford and BCA assays are common, fast, and rely on a dye- or copper-based color change to measure protein concentration spectrophotometrically.
Nitrogen-Based Methods: The Kjeldahl and Dumas methods quantify total nitrogen to estimate protein content, a standard approach in food analysis.
Standard Curve: Accurate quantification requires generating a standard curve using known protein concentrations to properly interpret the assay's results.
Interference: Be aware of common lab substances like detergents, reducing agents, and buffers, as they can significantly impact assay accuracy.
UV Absorbance: Direct measurement at 280 nm is quick and non-destructive but can be unreliable due to interference from nucleic acids and varying amino acid composition.
Accuracy vs. Speed: While some methods like Dumas offer high throughput, others like the Lowry assay provide greater sensitivity but at the cost of more time and complexity.

Core Principles of Protein Quantification

Accurately measuring the concentration of protein in a sample is a crucial, foundational step for many scientific and industrial applications. While the process may seem straightforward, a variety of factors influence the selection and execution of a specific assay. The choice is often a balance between speed, sensitivity, compatibility with sample components, and overall accuracy. Methods generally fall into a few broad categories, each leveraging different chemical properties of proteins to produce a measurable signal.

Considerations for Choosing a Method

Before beginning, analysts must weigh several key factors to select the most appropriate method for their specific sample:

Sensitivity: This refers to the lowest concentration of protein the assay can reliably detect. Highly sensitive assays are essential for samples with low protein content, such as dilute cell lysates.
Compatibility: Some assay reagents react with common laboratory substances like detergents (e.g., SDS, Triton X-100), reducing agents (e.g., DTT, β-mercaptoethanol), and certain buffers, leading to inaccurate results. It is crucial to choose an assay that is compatible with the sample's buffer composition.
Accuracy: The accuracy can be affected by the protein's specific amino acid composition. Colorimetric assays, for instance, may show variation in response to different proteins because they bind preferentially to certain amino acid side chains.
Speed and Throughput: For high-volume labs, automated methods like the Dumas assay are preferable due to their speed. In contrast, slower, multi-step assays like the Lowry might be used for higher precision in specific research contexts.

Colorimetric and Spectrophotometric Assays

Colorimetric assays are a common class of protein quantification methods that involve a chemical reaction producing a color change. The intensity of this color is proportional to the protein concentration and is measured using a spectrophotometer.

The Bradford Assay: This is one of the most widely used protein assays due to its speed and ease of use. It relies on the binding of Coomassie brilliant blue G-250 dye to protein, which causes a shift in the dye's absorption maximum from 465 nm to 595 nm. The absorption at 595 nm is then measured. While fast, its accuracy can be highly dependent on the protein's amino acid composition, especially basic and aromatic residues like arginine, lysine, and tryptophan.
The Bicinchoninic Acid (BCA) Assay: This two-step, copper-based colorimetric assay is more tolerant of detergents than the Bradford method. First, protein reduces Cu²⁺ to Cu⁺ under alkaline conditions. Second, bicinchoninic acid chelates with the Cu⁺ to form a stable purple complex that can be measured at 562 nm.
The Lowry Assay: An older but still valuable method, the Lowry assay is also a two-step, copper-based process. After the initial copper reduction, the Folin–Ciocalteu reagent is added, which is reduced by aromatic amino acids to produce a dark blue-green color. It is highly sensitive but more time-consuming and prone to interference than BCA.
Direct UV Absorbance at 280 nm: Some proteins can be quantified directly by measuring their UV absorbance at 280 nm, which is the wavelength strongly absorbed by the aromatic amino acids tryptophan and tyrosine. This method is fast and non-destructive, but its accuracy depends heavily on the protein's amino acid content and the presence of other UV-absorbing substances, such as nucleic acids.

Nitrogen-Based Quantification Methods

For applications in fields like food science, nitrogen-based methods provide a more general approach to determining crude protein content. These methods are based on the principle that, on average, protein contains a relatively consistent percentage of nitrogen.

The Kjeldahl Method: Developed in 1883, this is the traditional standard for measuring crude protein in food samples. It involves three steps: digestion of the sample in sulfuric acid to convert organic nitrogen to ammonium sulfate, distillation of ammonia after neutralizing with sodium hydroxide, and titration to quantify the captured ammonia. The nitrogen content is then multiplied by a conversion factor (typically 6.25) to estimate the total protein.
The Dumas Method: A modern, automated alternative to the Kjeldahl method. The sample is combusted at high temperatures, and the released nitrogen gas is measured using a thermal conductivity detector. The Dumas method is significantly faster, requires no hazardous chemicals, and is increasingly becoming the standard for food protein analysis.

Comparison of Protein Quantification Methods


Feature	Bradford Assay	BCA Assay	Lowry Assay	Kjeldahl Method	Dumas Method
Mechanism	Dye-binding to basic and aromatic amino acids.	Copper reduction by protein and chelation by BCA.	Copper reduction + reaction with Folin-Ciocalteu reagent.	Digestion to convert nitrogen to ammonia.	High-temp combustion to convert nitrogen to N₂ gas.
Sensitivity	High (20-2000 µg/ml)	High (20-2000 µg/ml)	Very High (1-1500 µg/ml)	High (mg/ml)	High (mg/ml)
Speed	Fast (10-15 mins)	Slower than Bradford (30-60 mins)	Slowest (45-60 mins)	Very slow (1-2 hours)	Very fast (<5 mins)
Interference	Detergents, basic proteins, some buffers.	Copper-chelating agents, reducing agents.	Many substances (e.g., detergents, Tris).	Non-protein nitrogen sources.	Non-protein nitrogen sources.
Cost	Low	Low to Moderate	Low	High (equipment)	High (equipment)
Safety	Safer than Lowry or Kjeldahl.	Safer than Lowry or Kjeldahl.	Hazardous chemicals.	Concentrated acid is a hazard.	Safer (no hazardous chemicals).

Step-by-Step Example: Performing a Bradford Assay

For a common lab quantification of protein concentration, the Bradford assay is an excellent choice. The general procedure is as follows:

Prepare a Standard Curve: Create a series of dilutions of a known protein standard, such as Bovine Serum Albumin (BSA), across the assay's linear range. This is critical for accurately determining the concentration of your unknown samples.
Dilute Dye Reagent: Dilute the Bradford assay dye reagent concentrate, typically 1:5 with distilled water.
Mix Samples and Standards: Combine the unknown protein samples and the known standards with the diluted dye reagent in separate test tubes or a microplate. A blank containing only buffer and dye is also necessary.
Incubate: Allow the reaction to proceed for the recommended time (e.g., 5-10 minutes) at room temperature.
Measure Absorbance: Use a spectrophotometer to measure the absorbance of each sample at 595 nm.
Analyze Data: Plot the absorbance readings of the known standards against their concentrations to create a standard curve. Use this curve to extrapolate the concentration of the unknown samples.

Conclusion

The ability to determine the amount of protein in a sample is a fundamental cornerstone of biochemical analysis. The optimal method is highly dependent on the specific application, available equipment, and the nature of the sample itself. While colorimetric assays like Bradford and BCA are popular for their speed and convenience in many research labs, nitrogen-based methods like Kjeldahl and Dumas offer standardized, robust results for industries like food science. For researchers, weighing the trade-offs between sensitivity, speed, and interference will lead to the most reliable and accurate quantification. For more information on commercial assay kits and methods, consider consulting Thermo Fisher Scientific's guide to protein assays.

Frequently Asked Questions

Amino acid analysis is often considered the most accurate method, as it directly measures the sum of all amino acid residues after protein hydrolysis, without interference from non-protein nitrogen. However, it is more complex and expensive than other methods.

The Bradford assay is a dye-binding method that is faster and more sensitive to basic and aromatic amino acids, but is inhibited by detergents. The BCA assay uses a copper-reduction reaction that is more tolerant of detergents and provides more uniform protein-to-protein response, though it is more sensitive to reducing agents.

Yes, but with limitations. It's a quick, non-destructive method for purified proteins, but accuracy is compromised in crude samples by the presence of nucleic acids or other UV-absorbing compounds.

The Lowry assay is sensitive to many common lab reagents, including reducing agents like DTT, EDTA, and Tris buffers, all of which can significantly interfere with the reaction and lead to inaccurate measurements.

In food science, the protein content is commonly determined by nitrogen-based methods, such as the Kjeldahl or Dumas methods, which measure the total nitrogen content. This is then converted to crude protein using a specific conversion factor.

A standard curve is a graph plotted with the absorbance values of protein solutions with known concentrations. It's crucial for quantification because it provides a reliable reference to interpolate and accurately calculate the concentration of unknown samples based on their absorbance.

The BCA assay is generally more compatible with samples containing detergents compared to the Bradford or Lowry methods. For samples with high detergent concentrations, specific detergent-compatible variations of the BCA assay are often used.