Bradford Assay

The traditional Bradford protein assay is widely used for quantifying protein concentrations. However, many of the commercial protocols are complicated. Amplite®™ Colorimetric Braford Protein Quantitation Assay Kit is a two-component and detergent-compatible assay to determine total protein concentrations. The assay is based on the same Coomassie Blue G-250 protein indicator as Bradford protein assay and provides comparable accuracy. Our proprietary formulation makes our kit much more convenient and rapid. The protein signal is monitored around 600 nm and assay is completed within 30 minutes. Amplite®™ Colorimetric Braford Protein Quantitation Assay Kit can be performed in a convenient 96-well microtiter-plate format and easily adapted to automation with no separation steps required.

Example protocol

AT A GLANCE

Protocol summary

Prepare 1X Bradford working solution (90 μL)
Add BSA standards or test samples (10 μL)
Incubate at room temperature for 5 - 15 minutes
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	TS	TS
BS6	BS6	...	...
BS5	BS5
BS4	BS4
BS3	BS3
BS2	BS2
BS1	BS1
BL	BL

Table 2. Reagent composition for each well

Well	Volume	Reagent
BS1-BS7	10 µL	Serial Dilutions (0.5 to 0.0078 mg/mL)
BL	10 µL	PBS
TS	10 µL	Test Samples

Prepare BSA standards (BS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 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.
Incubate the reaction at room temperature for 5 to 15 minutes.
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.

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 August 13, 2024

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