1. Microplate reader detection method:
1.1. Add 10μL of each BSA standard and protein sample into separate wells of the microplate.
Note: If the protein sample is rare, it can be diluted 5-10 times with PBS and multiplied by the dilution factor when calculating the protein concentration.
1.2. Mix BCA reagent A and reagent B to prepare working reagent in a 50:1 ratio. That is, 50mL of reagent A with 1mL of reagent B.
Note: Use the following formula to determine the total volume of working reagent needed. It is recommended to prepare 1-2 more wells of working reagent:
(BSA Standard + test sample) * (Replicates) * (BCA working reagent per sample) = total BCA working reagent volume.
Note: It is recommended to prepare the working reagent freshly before use. At room temperature, the working solution will gradually turn dark green, but it will not affect the quantification accuracy when it is used within 30 minutes.
1.3. Add 200μL BCA working reagent into each well and mix.
1.4. Seal the plate and incubate at room temperature for 5 minutes.
1.5. Measure the absorbance at 480 nm in a microplate reader.
Note: The incubation time may be extended if necessary, but should not exceed 15 minutes when measuring a wide range (0–10mg/mL) BSA Standard. This kit’s results are dynamic—the absorbance continuously increases, and the color development is relatively rapid. It is also possible to add 50μL of 1M HCl to end the reaction and extend the detection time, and complete the detection within 1 hour to minimize the impact on the results.
1.6. Subtract the blank OD480 from all absorbance values and plot the BSA standard curve: (OD480 on the Y-axis vs. BSA concentration on the X-axis). Calculate the protein concentration of each sample.
2. Spectrophotometric detection method:
2.1. Add 50μL of each BSA standard and protein sample into separate EP tubes.
Note: If the protein sample is rare, it can be diluted 5-10 times with PBS and multiplied by the dilution factor when calculating the protein concentration.
2.2. Mix BCA reagent A and reagent B to prepare working reagent in a 50:1 ratio. That is, 50mL of reagent A with 1mL of reagent B.
Note: Use the following formula to determine the total volume of working reagent needed. It is recommended to prepare 1-2 more wells of working reagent:
(BSA Standard + test sample) * (Replicates) * (BCA working reagent per sample) = total BCA working reagent volume.
Note: It is recommended to prepare the working reagent freshly before use. At room temperature, the working solution will gradually turn dark green, but it will not affect the quantification accuracy when it is used within 30 minutes.
2.3. Add 1000μL BCA working reagent into each well and mix.
2.4. Seal the lids and incubate at room temperature for 5 minutes.
2.5. Measure the absorbance at 480 nm using a spectrophotometer.
Note: The incubation time may be extended if necessary, but should not exceed 15 minutes when measuring a wide range (0–10mg/mL) BSA Standard. This kit’s results are dynamic—the absorbance continuously increases, and the color development is relatively rapid. It is also possible to add 250μL of 1M HCI to end the reaction and extend the detection time, and complete the detection within 1 hour to minimize the impact on the results.
2.6. Subtract the blank OD480 from all absorbance values and plot the BSA standard curve: (OD480 on the Y-axis vs. BSA concentration on the X-axis). Calculate the protein concentration of each sample.
Notes:
1. A series of protein standards with different concentrations should be dissolved and mixed at room temperature before use.
2. When reagent A and reagent B are mixed, turbidity may appear, and it will disappear after vortexing and mixing.
3. One microplate reader with a measuring wavelength of 480nm and a 96-well plate are required. If there is no microplate reader, a spectrophotometer can also be used for determination. However, when using spectrophotometer, the volumes of solution A, solution B, and the sample need to be adjusted proportionally based on the volume of the cup, and the samples that can be measured with each kit may be significantly reduced.
4. Incubate at room temperature (~25 °C) for 5 minutes before measurement. If color development is insufficient, the incubation time can be appropriately extended to 10-15 minutes. However, prolonged incubation time can cause the absorbance of high concentration protein standards to reach a plateau and cannot be used for calculating linear standard curves. The incubation temperature and time should be well handled.
5. The BCA method for determining protein concentration is not affected by the majority of chemical substances in the sample, and can be compatible with up to 5% SDS, 5% Triton X-100, 5% Tween 20/60/80 in the sample. The BCA method is affected by chelating agents and slightly higher concentrations of reducing agents, it is necessary to ensure that EDTA is below 10mM, no EGTA, dithiothreitol is below 1mM, and β-mercaptoethanol is below 1mM. If it is found that the sample diluent or lysate itself has a high background, please try the Bradford Protein Quantification Kit (catalog number: GK10027). You can also consider diluting samples with ultrapure water, Dialysis/desalination, ACETONE/TCA precipitation of proteins are used to remove interfering substances.
6. All reagents should be equilibrated to room temperature before use, and after use, each reagent should be immediately stored according to the conditions required by the reagent kit.
7. To obtain accurate results, perform color development and absorbance measurement for both standards and samples simultaneously, and draw a standard curve for each assay.
8. If the absorbance value at 480nm is not available, it can be measured at wavelength between 460-500nm, but the slope of the standard curve and the overall detection sensitivity will be slightly reduced (<10%).
