Biochemistry protein quantification · BEGINNER LESSON

Bradford assay

The Bradford assay is a color-based test used to measure how much protein is in a sample. It works by adding a Coomassie Brilliant Blue dye, then reading how strongly the sample absorbs light at 595 nm with a spectrophotometer.

AI-assisted lesson · Public sources · Updated 2026-10-12

What it is and what it measures

The Bradford assay is a colorimetric protein assay, meaning it estimates a substance by measuring color. It is used to quantify protein concentration in a sample. The sample is mixed with Coomassie Brilliant Blue dye, and the color change is read with a spectrophotometer, an instrument that measures how much light a sample absorbs at a chosen wavelength. In this assay, the key measurement is absorbance at 595 nm, which is compared with a known protein standard such as bovine serum albumin. This gives an estimate of protein amount rather than nitrogen content. [0,1,2]

[1] Melamine and Cyanuric Acid do not interfere with Bradford ... [2] Quantifying protein residues in APIs: Bradford assay ... - PMC [3] Evaluation of Colorimetric Assays for Analyzing Reductively ... - PMC

How the color change happens

The dye starts in a red form under acidic conditions. When it binds to proteins, it shifts toward a blue form, and the absorbance at 595 nm increases. The sources describe two main parts of this binding: electrostatic interaction, which is attraction between opposite charges, and hydrophobic interaction, which involves non-water-loving regions. Basic amino acids such as lysine and arginine, and in one source tryptophan, help the dye bind more strongly. This means different proteins can produce different signal strengths even if their amounts are similar. [0,1,2]

How the assay is used in practice

To run the test, a sample is mixed with Bradford reagent and then measured in a spectrophotometer. The reading is compared with a standard curve made from a known protein, so unknown samples can be estimated. The assay is described as rapid, simple, inexpensive, and widely used in molecular biology. It can also tolerate common biochemical reagents better than some other color tests. [0,1,2]

Limitations and sources of error

The Bradford assay is not equally reliable for every sample. The sources report weaker performance for some solid or poorly soluble materials, such as cat food, because incomplete solubility reduces the measured signal. Some compounds can also interfere, and a later source notes that color released by dissolved materials can distort the absorbance reading at 595 nm. Protein structure matters too: proteins with unusual amino acid composition or compact, hydrophobic structure may bind the dye less strongly. Chemical modification such as lysine methylation can slightly reduce the response, especially when many lysines are modified. [0,1,2]

SEE THE IDEA

Bradford assay workflow

01Protein sample

Unknown amount of protein in solution or prepared extract

02Coomassie dye

Red acidic dye that can bind proteins

03Blue complex

Protein-dye binding shifts the dye toward blue

04595 nm reading

Spectrophotometer measures absorbance to estimate protein

Protein binding changes the dye’s color, and the measured absorbance at 595 nm is compared with a standard to estimate protein amount.

A simple dye-binding assay: more protein binding usually means more blue signal, but sample composition can change the result.

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01 What does the Bradford assay measure directly?

02 A milk sample gives the expected Bradford reading, but a cat food sample gives a much lower reading because the proteins are not well dissolved. What is the best interpretation?

03 Why are protein standards needed in the Bradford assay?

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Sources and further reading

This general lesson was generated from public source material. It explains a concept; it does not interpret a private paper or provide medical advice.

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