SKU AR0146
Product nameBCA Protein Assay Kit(for 20-2000 μg/ml protein)
Pack size 1 kit
Applications Western blotting, protein expression assays, protein profiling and characterization, protein quantitation assays

List of Components

DescriptionQuantityVolume ContentsCatalog Number
BCA Reagent A1100mLcontaining sodium carbonate, sodium bicarbonate, bicinchoninic acid and sodium tartrate in 0.1M sodium hydroxideAR0146- A
BCA Reagent B15mLcontaining 4% cupric sulfateAR0146-B
Albumin (BSA) Standards120mL (2mg/mL) containing bovine serum albumin (BSA) at 2mg/mL in 0.9% saline and 0.05% sodium azideAR0146-C


Form Supplied BCA Reagent A and B: ready-to-use 1X solutions
BSA Standard: stock solution for preparing a series of standard protein dilutions
Assays per kit Tube procedure: 50 assays
Microplate procedure: 500 assays
Storage Upon receipt store at room temperature.
It is stable at room temperature for one year.
Note If either Reagent A or Reagent B precipitates upon shipping in cold weather or during long-term storage, dissolve precipitates by gently warming and stirring solution. Discard any kit reagent that shows discoloration or evidence of microbial contamination.
Assay Range 20 - 2000μg/mL
Equivalent Thermofisher (Product No. 23227), Assay Range: 20 - 2000μg/mL
Millipore Sigma (Product No. BCA1), Assay Range: 200 - 1000μg/mL
Compatibility with reagents Compatible with typical concentrations of most ionic and nonionic detergents
Incompatible with chelating agents, strong acids or bases, and reducing agents: interfere with the reduction and chelating reactions of the assay mechanism
Applications Western blotting, protein expression assays, protein profiling and characterization, protein quantitation assays
*Our Boster Guarantee covers the use of this product in the above tested applications.
Physical State of A+B Reagent mixture Light blue liquid
Reagent Color/Absorbance Blue / A562 nm
Activity Active in alkaline medium
Description BCA Protein Assay Kit is a ready-to-use detergent- compatible Western blot related total protein analysis reagent used for the quick determination of total protein concentration by measuring absorbance at 562 nm and comparing to a protein standard absorption vs. concentration curve, according to Smith. The protein quantification process can be finished in 45 minutes.
Cite This Product BCA Protein Assay Kit (Boster Biological Technology, Pleasanton CA, USA, Catalog # AR0146)

Assay Information

Assay Type Protein Quantitative Measurement
Sample Type Serum, Plasma, Cell culture extracts, Tissue Extracts
Technique Solution-based detection of spectral absorbance at 562 nm; Smith Protein assay
Detection Method Colorimetric/Spectroscopic
Equipment needed Test tubes or miscroplates; Spectrophotometer; Microplate Reader
Assay Purpose Measure protein concentration in solution
Incubation Requirements 30 min at 37°C
To be used with Lysates or homogenates, prepared with non-reducing lysis and extraction buffers: Mammal Tissue Protein Extraction Reagent, Mammal Cell Protein Extraction Reagent, RIPA Lysis Buffer, Mitochondria Extraction Reagent, Membrane Protein Extraction Kit, Cytoplasmic and Nuclear Extraction Kit, Bacterial Cell Protein Extraction Reagent

Assay Principle

The BCA Protein Assay is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantization of total protein. This method combines the well- known reduction of Cu2+ to Cu1+ by protein in an alkaline medium (the biuret reaction) with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu1+) using a unique reagent containing bicinchoninic acid. The purple-colored reaction product of this assay is formed by the chelation of two molecules of BCA with one cuprous ion. This water-soluble complex exhibits a strong absorbance at 562 nm that is nearly linear with increasing protein concentrations over a broad working range (20-2,000 μg/ml).
The BCA method is not a true end-point method; that is, the final color continues to develop. However, following incubation, the rate of continued color development is sufficiently slow to allow large numbers of samples to be assayed together. The macromolecular structure of protein, the number of peptide bonds and the presence of four particular amino acids (cysteine, cystine, tryptophan and tyrosine) are reported to be responsible for color formation with BCA. Studies with di-, tri- and tetrapeptides suggest that the extent of color formation caused by more than the mere sum of individual color producing functional groups.
Accordingly, protein concentrations generally are determined and reported with reference to standards of a common protein such as bovine serum albumin (BSA). A series of dilutions of known concentration are prepared from the protein and assayed alongside the unknown before the concentration of each unknown is determined based on the standard curve. If precise quantization of an unknown protein is required, it is advisable to select a protein standard that is similar in quality to the unknown; for example, a bovine gamma globulin (BGG) standard may be used when assaying immunoglobulin samples. Two assay procedures are presented. Of these, the Test Tube Procedure requires a larger volume (0.1 ml) of protein sample; however, because it uses a sample to working reagent ratio of 1:20 (v/v), the effect of interfering substances is minimized.

Additional Materials Required

• 37°C water bath or 37°C incubator

• Microplate reader and spectrophotometer capable of accurately measuring absorbance in the region of 540-595nm (562nm is the optimal wavelength)

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.

Assay Procedure

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

Tube NumberVolume of Diluent (μl)Volume of BSA (μl)Final BSA Concentration (μg/ml)
A0 μl900 μl of 2 mg/ml Stock2000 μg/ml
B100 μl300 μl of tube A1500 μg/ml
C300 μl300 μl of tube A 1000 μg/ml
D200 μl200 μl of tube B 750 μg/ml
E300 μl300 μl of tube C 500 μg/ml
F300 μl300 μl of tube E 250 μg/ml
G300 μl300 μl of tube F 125 μg/ml
H400 μl100 μl of tube G 25 μg/ml
I300 μl00 (blank)

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

Tube NumberVolume of Diluent(μl)Volume of BSA (μl)Final BSA Concentration (μg/ml)
A0 μl200 μl of 2 mg/ml Stock2000 μg/ml
B30 μl90 μl of tube A1500 μg/ml
C60 μl60 μl of tube A 1000 μg/ml
D60 μl60 μl of tube B 750 μg/ml
E60 μl60 μl of tube C 500 μg/ml
F60 μl60 μl of tube E 250 μg/ml
G60 μl60 μl of tube F 125 μg/ml
H100 μl25 μl of tube G 25 μg/ml
I60 μl00 (blank)

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.


ProblemPossible CauseSolution
No color in any tubesSample contains a copper chelating agentDialyze, desalt or dilute sample Increase copper concentration in working reagent (e.g., use 50:2, Reagent A:B)
Color of samples appears darker than expectedProtein concentration is too high
Sample contains lipids or lipoproteins
Dilute sample
Add 2% SDS to the sample to eliminate interference from lipids3
Need to measure color at a different wavelength Spectrophotometer or plate reader does not have 562nm filterColor may be measure at any wavelength between 540nm and 590nm, although the slope of standard curve and overall assay sensitivity will be reduced
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Customer Q&As

  • Q: What is the correct storage temperature of this kit? On the manual it says room temperature, but in the manual that we received in the past it said 4 degrees.
    A: We have modified the storage temperature of this kit as room temperature recently, but it can be stored at 4 degree as well. The BSA solution contains sodium azide, it is available at room temperature.
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