Introduction:

Currently, the most commonly used protein concentration detection methods in the world are: BCA Protein Assay Kit and Bradford Protein Assay Kit. Bradford assays are coomassie dye-binding assays for fast and simple protein quantification. Bradford protein assays are compatible with most salts, solvents, buffers, thiols, reducing substances, and metal chelating agents encountered in protein samples. The concentration of β-mercaptoethanol in the sample can be as high as 1 M and the concentration of DTT can be as high as 5 mM. However, it is significantly affected by high concentrations of detergents. Therefore, when using Bradford method for protein quantification, it is necessary to ensure that SDS is less than 0.01%, Triton X-100 is less than 0.05%, and Tween 20, 60, 80 is less than 0.015%. For protein quantification with high concentrations of detergents, the BCA Protein Assay Kit (Cat. #: EZPQ01) is recommended.

The Bradford Protein Assay Kit is mainly composed of Coomassie brilliant blue G250, buffer, etc. The detection speed is very fast. Generally, the detection of a small amount of samples can be completed in 10 minutes. The lower limit of the detection concentration reaches 25 μg/mL, the minimum detected protein amount reaches 0.5 μg, and the volume of the sample to be tested is 1-20 μL. There is a good linear relationship in the concentration range of 50~1000 μg/mL.

Product contents:

Storage: Store at room temperature, valid for 6 months. The protein standard should be frozen at -20°C after being prepared into solution.

Protocol:

1. Preparation of diluted BSA standards: Pipette 1 mL of Reagent C and add it to 20 mg of BSA powder (Reagent B), fully dissolve it to prepare a 20 mg/mL BSA standard solution. Take an appropriate amount of 20 mg/mL BSA standard solution and dilute to a final concentration of 500 μg/mL or the desired concentration. For example: take 25 μL of 20 mg/mL BSA standard solution, add 975 μL of diluent, mix well to prepare a 500 μg/mL diluted BSA standard solution. BSA standards can be diluted in 0.9% NaCl or PBS.
2. Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of the diluted BSA standard to the protein standard wells of the 96-well plate respectively, and add diluent to make up to 20 μL.
3. Add an appropriate volume of unknown sample to the sample well of the 96-well plate, and add diluent to 20 μL.
4. Add 200 µL of the Coomassie G250 Reagent (Reagent A) to each well and mix plate thoroughly and incubate at room temperature for 3~5 minutes.
5. Measure the absorbance at or near 595 nm on a plate reader. And the wavelength between 560~610 nm is also acceptable.
6. Prepare a standard curve by plotting the average 595 nm measurement for each BSA standard vs. its concentration in µg/mL. Use the standard curve to determine the protein concentration of each unknown sample.

Notes:

1. The same diluent should be used for the unknown protein sample and the BSA standard solution.
2. It is recommended to make a standard curve for each measurement.
3. If you do not have a microplate reader, you can also use a common spectrophotometer to measure.
4. For your safety and health, please wear a lab coat and disposable gloves for operation.
5. For research use only. Not for use in diagnostic procedures.