Introduction:

Currently, the most commonly used protein concentration detection methods in the world are: BCA Protein Assay Kit and Bradford Protein Assay Kit. The BCA Protein Assay is a popular method for colorimetric detection and quantitation of total protein. Compared with Bradford protein assay, BCA protein assay has an unique advantage——it has good compatibility with samples that contain up to 5% surfactants (detergents) such as SDS，Triton X-100, Tween 20 and Tween 80; and it is affected much less by protein compositional differences, providing greater protein-to-protein uniformity. However, BCA protein assay kit can be easily affected by chelating agents and high concentrations of reducing agents, so please try to make the concentration of EDTA≤10 mM, DTT≤1 mM, and 2-ME≤0.01%.

The BCA Protein Assay Kit has a good linear relationship in the concentration range of 50～1000 μg/mL, and the minimum detection amount is 25 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 D and add it to 20 mg of BSA powder (Reagent C), fully dissolve it to prepare a 20 mg/mL protein standard solution. Take an appropriate amount of 20 mg/mL protein standard and dilute to a final concentration of 500 μg/mL or the desired concentration.
2. Prepare BCA working solution by mixing 50 parts of Reagent A with 1 part BCA Reagent B (50:1, Reagent A: B). For the above example, combine 5 mL of Reagent A with 0.1 mL of Reagent B to prepare 5.1 mL of BCA working solution. BCA working solution is stable within 24 hours at room temperature.
3. 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.
4. Add an appropriate volume of unknown sample to the sample well of the 96-well plate, and add diluent to 20 μL.
5. Add 200 µL of the BCA working solution to each well and mix plate thoroughly and incubate at 37°C for 20~30 minutes.
6. Cool plate to RT. Measure the absorbance at or near 562 nm on a plate reader. And the wavelength between 540~595nm is also acceptable.
7. Prepare a standard curve by plotting the average 562 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. If the detection effect is found to be poor, it can be placed at room temperature for 2 hours or at 60°C for 30 minutes, and the color will continue to deepen with the extension of time and the increase of temperature.
3. When measuring the standard curve, if it is found that the absorbance or color does not change significantly with the increase of the standard concentration, the possible reason is that the sample contains substances that seriously interfere with the determination of protein concentration by BCA method.
4. It is recommended to make a standard curve for each measurement.
5. If you do not have a microplate reader, you can also use a common spectrophotometer to measure.
6. EDTA should not be contained in the samples in the BCA method, otherwise the test results will be affected.
7. For your safety and health, please wear a lab coat and disposable gloves for operation.
8. For research use only. Not for use in diagnostic procedures.