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Bradford Protein Assay Protocol

1. Key Features

The simplicity, speed, and high sensitivity of the Bradford assay have made it a preferred method in laboratories and companies worldwide, comparable in use to the BCA assay.


Assay Genie's Bradford Protein Reagent offers superior performance with the following characteristics:


High Sensitivity: Allows for the accurate measurement of proteins even at low concentrations.

Low Background: Provides a clear and precise signal, improving the reliability of results.

Significant Color Difference: The vivid blue color change upon binding makes the protein measurement intuitive and convenient to observe and quantify.

Jump to a section

keyboard_arrow_right   What is a Bradford Protein Assay?
keyboard_arrow_right   How does it work? 
keyboard_arrow_right   Kit Contents & Additional Material
keyboard_arrow_right   Protocol 
keyboard_arrow_right   Data Analysis
keyboard_arrow_right   Notes
keyboard_arrow_right   Validtion Data
keyboard_arrow_right   Technical Manual & Safety Datasheet

2.What is a Bradford Protein Assay

The Bradford assay is a rapid and highly sensitive method for quantifying protein concentration in a solution. It works on the principle of a dye-binding reaction involving Coomassie Brilliant Blue G-250 dye. 

3. How does it work? 

Dye Binding: In an acidic environment, the Coomassie Blue G-250 dye exists primarily in a cationic (red) and neutral (green) form.

Protein-Dye Complex Formation: When the dye binds to protein, primarily through ionic interactions with basic amino acid residues (like arginine, lysine, and histidine) and hydrophobic interactions, the dye is converted into its unprotonated, anionic (blue) form.

Absorbance Measurement: This protein-dye complex exhibits a maximum absorbance shift, with the highest absorption occurring at 595 nm.

Quantification: The increase in absorbance at 595 nm is nearly linear with increasing protein concentration over a wide working range, allowing for accurate quantification by comparing the sample's absorbance to a standard curve.

3.1. Kit Contents 

Included: Bradford Reagent 2ml | Store at Room Temperature (RT) for 1 Year.


Additional Materials Required: 

  • Microplate reader capable of measuring absorbance at 595 nm.
  • Bovine Serum Albumin (BSA).
  • Precision pipettes to deliver.
  • 96-well microplate.
  • Tubes to prepare standard or sample dilutions.
Figure 1. Illustration of Bradford Protein Assay Protocol.

3.2. 96-well Microplate Protocol

Step 1: Prepare a 5mg/ml BSA Stock using the same buffer in which your samples are dissolved (the assay buffer).

Step 2: Dilute the BSA Stock to create the following standards in your assay buffer: 0, 0.25, 0.5, 0.75, 1.0, and 1.5 mg/ml.

Step 3: Mark the appropriate wells as Standard or Sample. Add 10 μl of the diluted Standards to each Standard well, or add 10μl of your samples to the sample wells.

Step 4: Add 200 μl of Bradford Reagent to all wells and shake the plate for 30 seconds to mix.

Step 5: Incubate the plate at room temperature for 5-10 minutes. This allows the color-forming reaction to reach maximum stability.

Step 6: Measure the absorbance at 595 nm on a plate reader.

3.3. Data Analysis

  • Calculate Corrected Absorbance: Average the 595nm readings of your 0 mg/ml standard replicates (your blank). Subtract this average blank value from the 595nm reading of all individual standard and unknown sample replicates. 
  • Generate Standard Curve: Prepare a standard curve by plotting the Corrected 595nm measurement for each BSA standard versus its concentration in mg/ml (or convert to μg/ml if preferred for a wider range). 
  • Determine Unknown Concentration: Using the generated standard curve, determine the protein concentration estimate for each unknown sample by interpolation. 
  • Best Fit: If using curve-fitting software (like a microplate reader's program), a polynomial (quadratic or higher) or best-fit curve will provide more accurate results than a purely linear fit across the whole range. Note: If plotting results manually, a point-to-point curve is often preferable to a straight line connecting all standard points.
  • Apply Dilution Factor: If your samples were diluted prior to pipetting onto the plate, multiply the interpolated concentration by the original Dilution Factor to obtain the final protein concentration of the original stock sample.

3.4. Notes

  • Although reagents stored at 4°C rarely precipitate, a small amount of precipitation is acceptable and can be resolved by inverting the vial.
  • Bradford reagent testing is highly acidic. Please wear lab coat and gloves during assays for safety considerations. 
  • The Bradford Protein Reagent is largely intolerant to detergents. If lysis buffer contains detergents, then consider using BCA assay to quantify your protein levels.

4. Validation Data

The validation dataset for the Bradford Protein Assay demonstrates a clear and highly linear relationship between BSA concentration and absorbance at 595 nm. Standards ranging from 0 to 1.0 mg/ml produced absorbance values that increased proportionally across the entire measurement range, with the blank registering an absorbance of 0.368 and the highest concentration reaching 1.374. The resulting standard curve shows excellent linearity, confirming that the assay performs reliably within this concentration range. 


This strong correlation indicates that the Bradford assay is suitable for accurate quantification of protein samples, with minimal deviation between expected and observed values. The dataset supports the robustness, reproducibility, and analytical sensitivity of the assay.

Figure 2. Validation data and standard curve for the Bradford Protein Assay, demonstrating linear correlation between BSA concentration and absorbance at 595 nm across the 0–1.0 mg/ml range

Additional Resources