BCA Protein Assay Kit

A. Test Tube Procedure

1.Mix BCA Reagent A and BCA Reagent B at 50:1. i.e., mix 50 ml of BCA Reagent A with 1 ml BCA Reagent B.
Note: Use the following formula to determine the total volume of working reagent required. (# standards+ # unknowns) x (# replicates) x (volume of working reagent per sample)= total volume of working reagent required.
2.Follow Table 1 to prepare a fresh set of standards. (Dilute Albumin (BSA) Standards with 0.9% NaCl or PBS)

Table 1: Preparation of Albumin (BSA) Standards

3.Add 0.1 ml of each standard and protein samples into separate labeled test tubes.
4.Add 2 ml of BCA working reagent to each tube and mix well.
5.Incubate at 37°C for 30 minutes.
Note: Increasing the incubation time and temperature can increase the net 562 nm absorbance for each test and decreases both minimum detection level and test range of the kit.
6.Cool all tubes to room temperature (RT).
7.Set the wavelength of spectrophotometer at OD 562 nm. Calibrate the instrument to zero by using water. Subsequently, measure the absorbance of all samples within 10 minutes.
Note: Color development continues even after cooling to RT. However, the subsequent development at RT is too weak to produce significant error if all absorbance measurements are made within 10 minute.
8.Subtract OD562 of Blank from all readings.
9.Plot the BSA standard curve: OD562 (on Y axis) vs BSA Standard concentration (on X axis). Use the standard curve to determine the protein concentration of each unknown sample.

B. Microplate Procedure

1. Mix BCA Reagent A and BCA Reagent B at 50:1. i.e., mix 50 ml of BCA Reagent A with 1 ml BCA Reagent B.
Note: Use the following formula to determine the total volume of working reagent required. (# standards+ # unknowns) x (# replicates) x (volume of working reagent per sample)= total volume of working reagent required.
2.Follow Table 2 to prepare a fresh set of standards. (Dilute Albumin (BSA) Standards with 0.9% NaCl or PBS)

Table 2: Preparation of Albumin (BSA) Standards

3. Add 25 μl of each standard and protein samples into separate microplate wells.
4. Add 200 μl of BCA working reagent to each well and mix well.
5. Seal plates and incubate at 37°C for 30 minutes.
6. Cool plate to room temperature (RT).
7. Measure the absorbance at 562 nm on a plate reader within 10 minutes.
8. Subtract OD₅₆₂ of Blank from all readings. Plot the BSA standard curve: OD562 (on Y axis) vs BSA Standard concentration (on X axis). Use the standard curve to determine the protein concentration of each unknown sample.

Important product information

1. If this kit is received or stored cold, a precipitate may form in Reagent A or Reagent B. To dissolve the precipitate, warm the solution slowly at 37°C while mixing or microwave for a few seconds. Discard the kit if it is contaminated by bacteria
2. If interference caused by reducing substances or metal- chelating substances contained in the sample remains, Bradford Assay Kit is recommended.
3. It is recommended that the standard of different concentrations and samples be assayed in duplicate. Standard curve should be plotted for each assay.
4. Newly formed green turbidity will disappear after mixing Reagent A and Reagent B thoroughly. It will not affect the performance.
5. Assayed sample amount will be reduced while using spectrophotometer to detect protein concentration. When using 37° C incubator, prevent the influence of water evaporation.
6. Several ways for eliminating or minimizing the effects of interfering substances:
• Remove interfering substances by dialysis or gel filtration.
• Dilute the sample until substances no longer interfere.
• Since detergents of high concentrations also affect the results, precipitate the proteins in the sample with trichloroacetic acid (TCA).
7.Avoid using substances including reducing substances, chelating agents, strong acid and alkali since they interfere with protein estimation even at very low concentration.