UV/VIS Application Note

Bradford Protein Assay

Summary
Bradford assay is used to determine protein Solution BSA protein std, Purified water Final conc.
2 mg/mL [mL] [mL] [mg/mL]
concentration in biological samples. In this assay,
A 0.000 1.000 0.00
Brilliant Blue G and the protein forms a complex,
B 0.125 0.875 0.25
due to which a spectral shift occurs from brown
C 0.250 0.750 0.50
(465 nm) to blue (595 nm). Bovine serum
Albumin (BSA) is used as the protein standard in D 0.500 0.500 1.00

this assay. Samples are measured at a wavelength E 0.700 0.300 1.40

of 595 nm and a linear curve of absorbance F 0.350 0.650 0.70

versus concentration of standards is obtained.
Using this curve, the concentration of unknown
protein samples can be determined.
(Test sample)
Table1: Preparation of standard protein by serial dilution
Preparation of colorimetric standard S1- S6

Label 6 glass tubes as S1, S2, S3, S4, S5 and S6.

Sample and Reagent
 Protein Standard (BSA) Solution (2 mg/ml),
Sigma-Aldrich, Product No. 1001784649
 Bradford Reagent or Coomassie Brilliant Blue
G250. Sigma-Aldrich, Product No. B6916
Pipette 0.1 mL of each solution into the tubes
(solution A to S1, solution B to S2, etc.). Add 3.0 mL
of Coomassie blue dye to each tube and mix by
gentle shaking. Incubate all the test tube solutions at
room temperature for 5 to 10 min. Transfer the
incubated solution to a 1 cm disposable cuvette.
Blank measurement is performed using solution A
(S1) before standard measurement. Measure the
absorbance of the standards in increasing order of
concentration (A to E) followed by the sample
solution F (S6).

Measurement
Method parameters

Instruments and Accessories Method: Bio Quant

 UV7 Spectrophotometer (ME-30254726) Path length: 1.0 cm
 XP 205 Analytical balance (ME-11106027) No. of standards: 5
 Rainin pipettes Concentrations: 0.0, 0.25, 0.50, 1.00,
- 100 - 1000µL (ME-17011782)
1.40 mg/mL
- 500 – 5mL (ME-17011790)
No. of calibration curves: 1
 10 mL glass test tubes
 Disposable 10 mm pathlength cuvette Wavelength: 595
Background correction: None
Sample preparation Fit type 1: Linear
Dilution factor: 1.0
Preparation of working solution Calculation R1: Concentration
Prepare a series of protein standards concentration Unit: mg/mL
ranging 0.25 mg/mL – 1.4 mg/mL by serial
Note: this method is available as a template on the
dilution in a 10 mL glass test tube. Also prepare
UV5Nano and UV5Bio spectrophotometer.
dilutions of the unknown sample to be measured.

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 Acceptance criteria Conclusion
UV/VIS Application Note

The Bradford assay was successfully performed on the

2
Coefficient of determination R ≥ 0.98 UV7 spectrophotometer. The BSA standard curve was
linear up to 1.4 mg/mL. The protein sample
Recovery of test sample 0.70 ± 0.04 concentration is calculated automatically using the
standard curve. Specified acceptance criteria are met:
the coefficient of determination meets the requirements
and measurements are repeatable and comply as well.
Results
Reference
Concentration
Coefficient of Sigma Technical Bulletin – Bradford Reagent Catalog
Std of BSA Std. Absorbance @
determination Number B6916
solution Solution 595 nm
(R2)
[mg/mL]
Further information
S1 0.00 0.0009
www.mt.com/uv-vis
S2 0.25 0.0959

S3 0.50 0.1822 0.999

S4 1.00 0.3814

S5 1.40 0.5315

Fig. 1: Absorbance at 595 nm is plotted against concentration of

protein standards

Sample Conc.
Absorbance @ 595
(0.700 Recovery in mg/mL
nm
mg/mL)

S6 (1) 0.2707 0.72

S6 (2) 0.2711 0.72

S6 (3) 0.2689 0.71

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