Enhanced BCA Protein Assay Kit

Instruction

1. Preparation of BSA Standards
a. Add 1.2ml of BSA Preparation Solution into one tube of BSA Standard (30mg BSA) provided in this kit to make a 25mg/ml BSA standard solution after fully dissolving. Keep an appropriate amount for immediate use. Aliquot and store the rest at -20ºC for future use.
b. Dilute an appropriate amount of  25mg/ml BSA standard solution to a final concentration of 0.5mg/ml (e.g., mix 20μl of 25mg/ml BSA standard solution with 980μl of diluent). The standard should be diluted with the buffer used for sample preparation, or with the 0.9% NaCl or PBS for simplicity. The diluted 0.5mg/ml BSA standard can be stored at -20℃ for long term storage.
2. Preparation of BCA Working Solution
According to the number of assays, prepare an appropriate amount of BCA Working Solution by mixing BCA reagent A and BCA reagent B at a ratio of 50:1 (e.g., mix 5ml of BCA reagent A with 100µl of BCA reagent B) and mix well. The BCA Working Solution is stable for 24 hours at room temperature.
3. Determination of Protein Concentration
a. Pipette 0, 1, 2, 4, 8, 12, 16, and 20µl of the 0.5mg/ml BSA standard solution to the standard wells of the 96-well plate, and add the sample preparation buffer to a final volume of 20µl per well, which is equivalent to 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg/ml, respectively.
b. Add an appropriate volume of sample to the sample wells of the 96-well plate. If the sample is less than 20µl, add the sample preparation buffer to a final volume of 20µl per well. Record the sample volume used.
c. Add 200µl of BCA Working Solution to each well and incubate at 37ºC for 20-30 minutes.
Note: The reactions can also be incubated at room temperature for 2 hours, or at 60ºC for 30 minutes. The color intensity of the reactions will continue to increase over time, and the color development will be accelerated by the increase of temperature. If the protein concentration of a sample is low, we recommend incubating at a higher temperature or extending the incubation time appropriately.
d. Measure the absorbance at 562nm or other wavelength between 540-595nm with a microplate reader.
e. Calculate the protein concentration of the sample based on the standard curve and the sample volume used.

FAQ：
1. When measuring the standard curve, it was found that the absorbance or color intensity did not change significantly with the increase of standard concentrations.
The possible reason is that the sample preparation buffer contains substances that seriously interfere with the BCA Protein Assay. For a detailed information about the substances that cause interference with the BCA Protein Assay, please refer to our website at:
http://www.beyotime.com/Compatibility Chart For BCA Kit.pdf
2. Do I need to make a standard curve for every measurement?
It is recommended to make a standard curve for every measurement, because the color intensity of BCA reactions will increase over time and the color development is related to the temperature. Unless the time and temperature of the assay can be precisely controlled, a standard curve should be plotted for every measurement when accurate quantification is desired.