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Bradford Protein Assay

Product key features

  • Visible detection: Measures absorbance at 595 nm with a clear color shift from brown to blue.
  • Extended dynamic range: 595/460 nm ratio expands linear detection range up to 10-fold.
  • Applications: Ideal for quantifying total protein in biochemical and cell-based assays.
 

Product description

The Amplite® Colorimetric Bradford Protein Quantitation Assay Kit is a colorimetric assay that quantifies protein concentration by measuring the absorbance shift of Coomassie Brilliant Blue G-250 dye from brown to blue. In acidic conditions, the dye exists in a reddish-brown cationic form (maximum absorbance at 470 nm). Upon binding to proteins, it forms strong, noncovalent complexes and shifts to a blue anionic form with a peak absorbance at 595 nm. The increase in absorbance at 595 nm is directly proportional to the protein concentration in solution.

Unlike UV-based 280 nm measurements that rely on aromatic amino acids, the Bradford method is based on interactions with basic amino acids such as arginine, lysine, and histidine. This allows for visible-spectrum detection and even visual inspection of protein content. While the traditional linear range is limited to ~10 µg/mL due to spectral overlap, using a dual-wavelength ratio (595/460 nm) significantly extends the assay’s dynamic range, improving accuracy at higher protein concentrations.

Absorbance shift of Coomassie Brilliant Blue with increasing protein concentration

Example protocol

AT A GLANCE

Protocol summary
  1. Prepare 1X Bradford working solution (90 μL)

  2. Add BSA standards or test samples (10 μL)

  3. Incubate at room temperature for 5 - 15 minutes
  4. Measure the absorbance at 595 nm and 460 nm, calculate the ratio of A595/A460 nm

Important

Thaw all the kit components at room temperature before use.

PREPARATION OF STANDARD SOLUTIONS

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/11118

BSA Standard Solution
Add 50 µL of 1 mg/mL BSA Standard (Component C) to 50 µL of ddH2O (not provided) to generate 0.5 mg/mL BSA standard solution (BS7). Then perform 1:2 serial dilutions in ddH2O to get serially diluted BSA standards BS6 - BS1. Note: It is necessary to create a standard curve for each assay.

PREPARATION OF WORKING SOLUTION

Bradford working solution (1X)

Prepare 1X Bradford working solution by diluting 1 part Bradford Assay Solution (Component A) to 4 parts of ddH2O.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of BSA standards and test samples in a clear bottom 96-well microplate. BS= BSA Standards (BS1 - BS7, 0.5 to 0.0078 mg/mL); BL=Blank Control; TS=Test Samples

BS7

BS7

TSTS

BS6

BS6

......

BS5

BS5

  

BS4

BS4  

BS3

BS3

  

BS2

BS2

  

BS1

BS1

  

BL

BL  

Table 2. Reagent composition for each well

WellVolumeReagent

BS1-BS7

10 µL

Serial Dilutions (0.5 to 0.0078 mg/mL)

BL

10 µL

PBS
TS

10 µL

Test Samples
  1. Prepare BSA standards (BS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2.

  2. Add 90 µL of 1X Bradford working solution to each well of BSA standard, blank control, and test samples to make the total assay volume of 100 µL/well.

  3. Incubate the reaction at room temperature for 5 to 15 minutes.
  4. Measure the absorbance with an absorbance microplate reader at OD 595 nm and 460 nm, and calculate the ratio of A595/A460 nm.

    Note: Please read 595 nm only if no 460 nm is available for the instrument.

Bradford assay calculator

To calculate the concentration of a protein sample using the Bradford assay method, a standard curve must first be generated. This can be done by plotting the absorbances of a known set of protein solutions against their concentrations. Typically, BSA is used, with the solutions created using a serial dilution.
 

Instructions

Given a set of absorbance and concentration values, this calculator will produce the best fit regression line to use as a standard curve. Please provide the concentration values for the known protein standards. Then provide either the 595 nm absorbance values, or both 595 nm and 460 nm absorbance values (if using ratiometric calculation method), in the fields below.

Once data has been processed, select the column of absorbance values that corresponds with the protein standards. Clicking "calculate" will generate the standard curve. A table will be provided that indicates the concentrations of all protein samples, based on the absorbance values provided.

Begin Calculation

References

View all 50 references: Citation Explorer
The Effects of a Concentrated Surfactant Gel on Biofilm EPS.
Authors: Salisbury, Anne-Marie and Chen, Rui and Mullin, Marc and Foulkes, Lauren and Percival, Steven L
Journal: Surgical technology international (2020): 31-35
[Preliminary Investigation on Difference of Protein Compositions Between DC2.4 Cells and Their Derived Exosomes by nanoLC-MS/MS].
Authors: Lin, Qing and Li, Yan and Qu, Meng-Ke and Liu, Xing and Zhang, Zhi-Rong and Zhang, Ling
Journal: Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition (2020): 81-86
The effect of Chinese wild blueberry fractions on the growth and membrane integrity of various foodborne pathogens.
Authors: Zhou, Tong-Tong and Wei, Cai-Hong and Lan, Wei-Qing and Zhao, Yong and Pan, Ying-Jie and Sun, Xiao-Hong and Wu, Vivian C H
Journal: Journal of food science (2020): 1513-1522
Combined Fluorimetric Caspase-3/7 Assay and Bradford Protein Determination for Assessment of Polycation-Mediated Cytotoxicity.
Authors: Larsen, Anna K and Hall, Arnaldur and Lundsgart, Henrik and Moghimi, S Moein
Journal: Methods in molecular biology (Clifton, N.J.) (2019): 301-311
Pilose Antler Extracts (PAEs) Protect against Neurodegeneration in 6-OHDA-Induced Parkinson's Disease Rat Models.
Authors: Li, Chaohua and Sun, Yanan and Yang, Weifeng and Ma, Shuhua and Zhang, Lili and Zhao, Jing and Zhao, Xin and Wang, Yi
Journal: Evidence-based complementary and alternative medicine : eCAM (2019): 7276407
Page updated on January 31, 2025

Ordering information

Price
Unit size
1000 Tests
5000 Tests
Catalog Number
1111811119
Quantity
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Additional ordering information

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Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501

Platform

Absorbance microplate reader

Absorbance595 nm, 460 nm
Recommended plateClear bottom

Components

BSA dose responses were measured with Amplite® Colorimetric Bradford Protein Quantitation Assay Kit using a clear bottom 96-well plate. A) Detect with A595/A460nm. B) Detect with A595 nm if A460 nm is not available.
BSA dose responses were measured with Amplite® Colorimetric Bradford Protein Quantitation Assay Kit using a clear bottom 96-well plate. A) Detect with A595/A460nm. B) Detect with A595 nm if A460 nm is not available.
BSA dose responses were measured with Amplite® Colorimetric Bradford Protein Quantitation Assay Kit using a clear bottom 96-well plate. A) Detect with A595/A460nm. B) Detect with A595 nm if A460 nm is not available.