Bradford Assay

The Bradford Assay is a colorimetric (relying on color change) protein quantification method. At the heart of this assay is the Coomassie Brilliant Blue G-250 dye. In its unbound form, this dye is reddish-brown and absorbs light at 465 nm. However, when it binds to protein, primarily through arginine and lysine residues, the dye undergoes a color change to blue and its maximum absorbance shifts to 595 nm. This change in color and absorbance is directly proportional to the protein concentration, allowing for quantification.

Workflows requiring a Bradford Assay

Protein Purification

After each purification step, it’s essential to determine the concentration of the purified protein to assess the yield and purity. The Bradford Method provides a quick and reliable method to quantify the protein in these fractions. It helps researchers decide which fractions to pool and aids in determining the efficiency and success of the purification process.

Cell Lysate Preparation

Before using the lysate for downstream applications, it’s crucial to know its protein concentration. This ensures that equal amounts of protein are used across samples, allowing for consistent and comparable results. The Bradford Assay is commonly used to determine the total protein content in these lysates.

Sample Preparation for SDS-PAGE

For accurate results, it’s essential that each well in an SDS-PAGE gel receives an equal amount of protein. This ensures that when comparing bands across lanes, any differences observed are due to the experimental conditions and not unequal loading. The Bradford Assay is used to adjust the protein concentrations of samples so that equal amounts of protein are loaded into each well.

Bradford Assay Methods

Standard Curve Method

This is the most common method and involves creating a calibration curve using known concentrations of a standard protein, typically Bovine Serum Albumin (BSA) or gamma globulin.

Procedure:

1. Prepare a series of dilutions of the standard protein to cover the expected range of protein concentrations in your samples.
2. Add Bradford reagent to each standard dilution and incubate for a short period (usually 5-10 minutes).
3. Measure the absorbance of each standard at 595 nm using a spectrophotometer.
4. Plot the absorbance values against the known protein concentrations to generate a standard curve.
5. Measure the absorbance of your unknown samples and determine their protein concentrations using the standard curve.

Single-Point Method

A quicker alternative to the standard curve method. It involves estimating the protein concentration of an unknown sample using a single known concentration of a standard protein.

Procedure:

1. Prepare a single concentration of the standard protein that’s within the expected range of your samples.
2. Add Bradford reagent to the standard and your unknown samples.
3. Measure the absorbance of both the standard and the unknown samples.
4. Calculate the protein concentration of the unknown samples using the ratio of the absorbances and the known concentration of the standard.

Microplate Method

This method adapts the Bradford Assay for high-throughput analysis using microplates, allowing for the simultaneous measurement of multiple samples.

Procedure:

1. Pipette standards and unknown samples into the wells of a microplate.
2. Add Bradford reagent to each well.
3. Incubate for the required time.
4. Use a microplate reader to measure the absorbance of each well at 595 nm.
5. If using the standard curve method in a microplate format, plot the absorbance values of the standards against their known concentrations to generate a calibration curve. Determine the protein concentrations of the unknown samples using this curve.

Benefits of Automation over Manual Pipetting for a Bradford Assay:

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