The Bradford method is a colorimetric (relying on color change) protein quantification method. At the heart of the method is Coomassie Brilliant Blue G-250 dye. This dye is reddish-brown in its unbound state and absorbs light at a wavelength of 465 nm. However, when it binds to proteins primarily through arginine and lysine residues, the color of the dye changes to blue and the maximum absorbance changes to 595 nm. This change in color and absorbance is proportional to protein concentration and can be used for quantitation.

Workflow requiring Bradford method

Protein purification

After each purification step, determining the concentration of the purified protein is critical to assess yield and purity. The Bradford method provides a fast and reliable method for protein quantification, helping researchers determine which components need to be combined and helping to evaluate the efficiency and success rate of the purification process.

Cell lysate preparation

Before using a lysate for downstream applications, it is critical to know its protein concentration. This ensures that equal amounts of protein are used in different samples, resulting in consistent and comparable results. The Bradford method is commonly used to determine the total protein content in these lysates.

SDS-PAGE sample preparation

To obtain accurate results, each well in an SDS-PAGE gel must contain an equal amount of protein. This ensures that when comparing bands across lanes, any differences observed are due to experimental conditions rather than uneven loading. The Bradford method can be used to adjust the protein concentration of a sample so that the amount of protein in each well is equal.

Bradford test method

Standard curve method

This is the most common method and involves developing a calibration curve using a standard protein of known concentration, usually bovine serum albumin (BSA) or gamma globulin.

1. Prepare a series of standard protein dilutions to cover the expected range of protein concentrations in the sample.
2. Add Bradford's reagent to each standard dilution and incubate for a short period of time (usually 5-10 minutes).
3. Measure the absorbance of each standard using a spectrophotometer at a wavelength of 595 nm.
4. Compare the absorbance values ??to known protein concentrations to generate a standard curve.
5. Measure the absorbance of the unknown sample and determine its protein concentration based on the standard curve.

Single point method

A quick alternative to the standard curve method. It uses a single standard protein of known concentration to estimate the protein concentration of an unknown sample.

1. Prepare a single standard protein at a concentration within the range expected for your sample.
2. Add Bradford reagent to standard samples and unknown samples.
3. Measure the absorbance of standard and unknown samples.
4. Calculate the protein concentration of the unknown sample using the ratio of the absorbance to the concentration of a known standard.

Microplate method

This method modifies the Bradford method into a high-throughput analysis using microplates that can measure multiple samples simultaneously.

1. Transfer standards and unknown samples into the wells of a microplate.
2. Add Bradford reagent to each well.
3. Time required for cultivation.
4. Measure the absorbance of each well at a wavelength of 595 nm using a microplate reader.
5. If the standard curve method is used in a microplate format, the absorbance values ??of the standards are compared to their known concentrations to generate a calibration curve. Use this curve to determine the protein concentration of an unknown sample.

Advantages of the automated Bradford method compared to manual pipetting:

• Consistency and repeatability: Automation reduces human error, making results more consistent.
• High-throughput: Automated systems can process multiple samples simultaneously, increasing efficiency.
• Reduced risk of contamination: Automation minimizes the chance of cross-contamination between samples.
• Data logging: Automated systems often come with software that logs data, making it easier to track and analyze results.