Manufactured by Aladdin, the Bradford Protein Assay Kit is developed based on the Bradford method, one of the two most widely used protein quantification approaches. It enables rapid, stable, and highly sensitive protein concentration measurement, with excellent linearity of detection data.
Assay Principle of the Bradford Method, In an acidic medium, Coomassie Brilliant Blue G-250 binds to basic and aromatic amino acids in proteins, especially arginine. This binding turns the solution blue and shifts the maximum absorbance peak of the dye from 465 nm to 595 nm. The degree of color change is proportional to protein concentration. Therefore, the protein concentration in a sample solution can be quantified by measuring absorbance at 595 nm.
B774074
| Component
| 1000T | Storage
|
B774074A
| Bradford Protein Assay Dye Reagent
| 100 mL×2 | 2-8℃ |
B774074B
| Protein Standard (5 mg/ml BSA)
| 1 mL | -20℃ |
Product Advantages:
1. Ultra-fast detection: Assays for 10–80 samples can be completed in less than 10 minutes.
2. High sensitivity: The lower limit of detectable concentration is 25 μg/mL, with a minimum detectable protein mass of 0.5 μg; the recommended sample loading volume ranges from 1 to 20 μL.
3. Excellent linearity within the concentration range of 50–1000 μg/mL.
4. The Bradford protein quantification assay is resistant to interference from most chemical substances in samples, but sensitive to elevated detergent concentrations. Ensure sample detergent levels comply with the limits below: SDS < 0.01%, Triton X-100 < 0.05%, Tween 20 / 60 / 80 < 0.015%. For samples containing detergents, we recommend our Ready-to-Use BCA Protein Quantitation Kit (Cat. No. B736762) or Detergent-Compatible Bradford Protein Quantitation Kit (Cat. No. B774078).
5. Each kit supports up to 1000 independent assays.
Instructions for Use:
I. Preparation of Protein Standards
Dilution Scheme for Standard Test Tube & Standard Microplate Assays (Detection range: 100–1500 μg/mL)
Tube No.
| Volume of Diluent
| Volume of BSA Standard
| Final Concentration
|
| A | 600 μl
| 400 μl (Taken from the 5 mg/ml tube)
| 2000 μg/ml
|
| B | 125 μl
| 375 μl (Taken from tube A)
| 1500 μg/ml
|
| C | 250 μl
| 250 μl (Taken from tube A)
| 1000 μg/ml
|
| D | 175 μl
| 175 μl (Taken from tube B)
| 750 μg/ml
|
| E | 325 μl
| 325 μl (Taken from tube C)
| 500 μg/ml
|
| F | 325 μl
| 325 μl (Taken from tube E)
| 250 μg/ml
|
| G | 325 μl
| 325 μl (Taken from tube F)
| 125 μg/ml
|
| H | 400 μl
| 100 μl (Taken from tube G)
| 25 μg/ml
|
| I | 400 μl
| 0
| 0 μg/ml (Blank well) |
Dilution Scheme for Micro Test Tube & Microplate Assays (Detection range: 1–25 μg/mL)
Tube No.
| Volume of Diluent
| Volume of BSA Standard
| Final Concentration
| | A | 3980 μl
| 20 μl (Taken from the 5 mg/ml tube)
| 25 μg/ml
| | B | 800 μl
| 3200 μl (Taken from tube A)
| 20 μg/ml
| | C | 1000 μl
| 3000 μl (Taken from tube B)
| 15 μg/ml
| | D | 1000 μl
| 2000 μl (Taken from tube C)
| 10 μg/ml
| | E | 1500 μl
| 1500 μl (Taken from tube D)
| 5 μg/ml
| | F | 1500 μl
| 1500 μl (Taken from tube E)
| 2.5 μg/ml
| | G | 4000 μl
| 0
| 0 μg/ml (Blank well)
|
|
II. Protein Concentration Determination
Test Tube Assay
A. Standard Test Tube Assay (Detection Range: 100–1500 μg/mL)
1. Transfer 0.05 mL of each standard and unknown sample into correspondingly labeled test tubes.
2. Add 1.5 mL of Bradford protein assay dye reagent to each tube and mix thoroughly.
3. Optional guideline: Incubate samples at room temperature for 10 minutes for most applications.
4. Set the spectrophotometer wavelength to 595 nm and zero the instrument, then measure the absorbance of all samples.
5. Subtract the average absorbance value of the blank control at 595 nm from the readings of all standards and unknown samples.
6. Generate a standard curve: Correct the absorbance reading of each BSA standard at its corresponding concentration using the average blank value. Calculate the protein concentration of each unknown sample based on this standard curve.
B. Micro Test Tube Assay (Detection Range: 1–25 μg/mL)
1. Transfer 1.0 mL of each standard and unknown sample into correspondingly labeled test tubes.
2. Add 1.0 mL of Bradford protein assay dye reagent to each tube and mix thoroughly.
3. Optional guideline: Incubate samples at room temperature for 10 minutes for most applications.
4. Set the spectrophotometer wavelength to 595 nm and zero the instrument, then measure the absorbance of all samples.
5. Subtract the average absorbance value of the blank control at 595 nm from the readings of all standards and unknown samples.
6. Generate a standard curve: Correct the absorbance reading of each BSA standard at its corresponding concentration using the average blank value. Calculate the protein concentration of each unknown sample based on this standard curve.
Microplate Assay
A. Standard Microplate Assay (Detection Range: 100–1500 μg/mL)
1. Pipette 10 μL of each standard and unknown sample into correspondingly labeled wells of the microplate.
2. Add 300 μL Bradford protein assay dye reagent to each well, mix and shake vigorously for 30 seconds.
3. Stop shaking. For most samples, incubate the plate at room temperature for 10 minutes.
4. Measure the absorbance at 595 nm using a microplate reader.
5. Subtract the average absorbance value of blank wells at 595 nm from the readings of all standards and unknown samples.
6. Generate a standard curve: Correct the absorbance value of each BSA standard at its corresponding concentration with the average blank reading. Calculate the protein concentration of each unknown sample from the standard curve.
B. Micro Microplate Assay (Detection Range: 1–25 μg/mL)
1. Pipette 150 μL of each standard and unknown sample into correspondingly labeled wells of the microplate.
2. Add 150 μL Bradford protein assay dye reagent to each well, mix and shake vigorously for 30 seconds.
3. Stop shaking. For most samples, incubate the plate at room temperature for 10 minutes.
4. Measure the absorbance at 595 nm using a microplate reader.
5. Subtract the average absorbance value of blank wells at 595 nm from the readings of all standards and unknown samples.
6. Generate a standard curve: Correct the absorbance value of each BSA standard at its corresponding concentration with the average blank reading. Calculate the protein concentration of each unknown sample from the standard curve.
Precautions:
1. Equilibrate the Coomassie G-250 dye reagent to room temperature before use to improve detection sensitivity. Invert the bottle 3–5 times and mix thoroughly prior to each use.
2. Fully dissolve the BSA protein standard stock solution and mix well before serially diluting it to prepare standards of different concentrations.
3. For accurate measurements, record absorbance values within 5–20 minutes after reagent addition, as the color remains most stable during this window.
4. Most cations including K⁺, Na⁺, Mg²⁺, ammonium sulfate ((NH₄)₂SO₄), and ethanol do not interfere with the assay. However, high concentrations of detergents such as Triton X-100 and SDS will severely disrupt detection results.
5. For personal safety and health, wear a lab coat and disposable gloves during all operations.
G2601311