BCA Assay Protocol: How to Measure Protein Concentration
BCA Assay Protocol for Protein Quantification
Learn how the BCA protein assay works, prepare a reliable standard curve, calculate unknown protein concentrations, control chemical interference and use the AffiASSAY® BCA Protein Quantification Kit.
What is a BCA assay?
A BCA assay is a colorimetric method for measuring total protein concentration. Protein reduces Cu²⁺ to Cu⁺ under alkaline conditions. Bicinchoninic acid then chelates Cu⁺ to form a purple complex. Its absorbance is measured near 562 nm and compared with a protein standard curve to determine unknown concentrations. 1 2
Protein concentration affects Western blot loading, immunoprecipitation, enzyme assays, protein purification, ELISA development and cell- or tissue-lysate normalisation. Reliable quantification is therefore required before many downstream experiments can be compared.
BCA means bicinchoninic acid. Smith and colleagues introduced the method in 1985 as a copper-based protein assay combining the biuret reaction with sensitive detection of cuprous ions. 1
The method is widely used because it offers broad practical measurement, relatively low protein-to-protein variation and compatibility with many common detergents. Reducing agents, metal chelators and certain complex sample matrices can nevertheless interfere. 1 3
How does the BCA assay work?
The assay combines copper reduction with selective detection of Cu⁺.
Protein reacts with alkaline copper
Peptide bonds and certain amino-acid residues reduce Cu²⁺ to Cu⁺.
BCA binds the reduced copper
Two BCA molecules chelate one Cu⁺ ion to form a purple complex.
Absorbance is compared with standards
Blank-corrected A562 values are interpolated from a BSA calibration curve.
Why do proteins sometimes give different colour responses?
Signal depends on peptide bonds and residues including cysteine or cystine, tyrosine and tryptophan. Equal masses of two different proteins may therefore produce slightly different absorbance. BSA is a convenient standard, but a protein-matched standard may improve absolute accuracy. 1 2
Is the BCA reaction a true endpoint?
No. Colour can continue developing after incubation. Standards and samples should be incubated, cooled and read using consistent timing. 2
AffiASSAY® BCA Protein Quantification Kit
A colorimetric research kit for determining protein concentration at 562 nm.
Current AffiGEN product information
AffiGEN describes protein reduction of Cu²⁺ to Cu⁺ under alkaline conditions, followed by BCA colour formation and measurement at A562. The kit is for research use only. 6
Step-by-step BCA protein assay workflow
The scientific workflow below must be adapted to the current AffiASSAY® manual.
Check buffer compatibility
Identify detergents, reducers, chelators, lipids and coloured components.
Prepare BSA standards
Create a concentration series covering the expected unknowns.
Prepare working reagent
Mix reagents A and B according to the current instructions.
Add blank, standards and samples
Use consistent volumes and technical replicates.
Add reagent and mix
Ensure equal contact while avoiding bubbles and contamination.
Incubate uniformly
Apply identical temperature and time to every assay position.
Cool and read
Measure absorbance near 562 nm within a consistent time window.
Subtract the blank
Correct all standards and unknowns using the mean blank value.
Calculate concentration
Interpolate within the curve and apply the full sample dilution factor.
How should standards be prepared?
Prepare known BSA concentrations using, whenever possible, the same diluent as the samples. Buffer matching reduces differences caused by salts, detergents and pH. 2 1
Should samples be tested in duplicate?
Technical duplicates are commonly used to detect pipetting or well-specific errors. More replicates may be needed during method development or near the lower measurement range.
Can another wavelength be used?
The purple BCA-Cu⁺ complex is normally measured near 562 nm. Measurements away from the absorbance maximum can reduce sensitivity, so the wavelength specified in the AffiASSAY® instructions should be used. 1
How to calculate protein concentration from a BCA assay
Blank-correct, fit the standards, interpolate and apply pre-assay dilution.
Build the BCA standard curve
Plot concentration on X and blank-corrected A562 on Y.
Subtract the mean reagent blank.
Choose a model that represents the standards.
Do not extrapolate outside the reliable range.
Apply every dilution made before the assay.
Protein concentration calculation
When the accepted standard curve is linear and follows y = mx + b, the protein concentration can be calculated by rearranging the equation.
Example: for y = 0.00072x + 0.015, a blank-corrected absorbance of 0.375 corresponds to 500 µg/mL. When the sample was diluted fourfold before testing, the original concentration is 2,000 µg/mL.
Use the curve-fitting model accepted for the measured standards. Do not force a linear equation when the calibration data show meaningful curvature.
Linear calculation example
For y = 0.00072x + 0.015 and sample absorbance 0.375, the diluted concentration is 500 µg/mL. A fourfold dilution gives an original concentration of 2,000 µg/mL, or 2 mg/mL.
When should a polynomial fit be used?
A broad BCA standard curve may show curvature. Use a curve-fitting model that accurately represents the measured standards and validate the model before calculating unknown concentrations. 2 1
Should reagent dilution be included in the dilution factor?
Usually no, because standards and unknown samples receive the same assay reagent. Apply the dilution factor used on the original sample before it was placed in the assay, unless the validated analysis procedure specifies otherwise. 1
What interferes with a BCA protein assay?
BCA tolerates many detergents, but not every sample component.
Reducing agents
They can reduce copper without protein and produce falsely high results.
- DTT
- β-mercaptoethanol
- TCEP at incompatible levels
- Ascorbate and related reducing compounds
Copper chelators
They bind copper and can weaken colour formation.
- EDTA
- EGTA
- Citrate at incompatible levels
- Other strong metal-binding compounds
Lipids and complex matrices
Turbidity, lipids or extraction components can affect colour or optics.
- Lipid-rich lysates
- Turbid tissue extracts
- Particulate material
- Strongly coloured components
Extreme buffer conditions
Highly buffered or chemically unusual samples can disrupt alkaline conditions.
- Very high buffer concentrations
- Incompatible pH
- High salt or additive combinations
- Additive interference
AffiGEN states that common concentrations of SDS, Triton X-100 and Tween-20 generally do not affect detection, while EDTA, EGTA, DTT, mercaptoethanol and lipids can interfere. Confirm compatibility for the exact final buffer. 6 3 4 5
How can interference be reduced?
Options include sample dilution, buffer-matched standards, desalting, dialysis, buffer exchange or protein precipitation. Buffer matching can reduce ordinary matrix differences, but it cannot reliably correct severe copper reduction or chelation.
Common BCA assay problems
Begin with the blank, standards, buffer and timing.
| Observation | Possible cause | Action |
|---|---|---|
| High blank | Reducing agent, contamination or over-incubation. | Prepare fresh blanks, verify reagents and review the buffer. |
| No or weak colour | Chelator, omitted reagent or insufficient incubation. | Check EDTA/EGTA, reagent addition and current instructions. |
| Flat standard curve | Incorrect standards, wavelength or incubation. | Prepare fresh standards and verify A562 conditions. |
| High replicate variation | Pipetting, bubbles, mixing or edge effects. | Calibrate pipettes, mix uniformly and remove bubbles. |
| Sample above range | Protein concentration exceeds the curve. | Dilute, repeat and apply the total dilution factor. |
| Sample below range | Concentration is too low. | Concentrate the sample or use a more sensitive method. |
| Result too high | Reducing interference, wrong blank or dilution factor. | Test buffer alone and audit the calculation. |
| Result too low | Chelation, precipitation or under-incubation. | Review composition, solubility and incubation. |
What is a BCA protein assay used for?
Total protein measurement supports normalisation and defined protein input.
Western blot
Load comparable total protein mass in each lane.
Cell and tissue lysates
Normalise biomarkers or enzyme results to total protein.
Protein purification
Track concentration across fractions and final formulation.
ELISA development
Quantify antigens, standards and conjugate preparations.
Enzyme assays
Calculate activity per unit of total protein.
Immunoprecipitation
Standardise lysate protein input before capture.
Western blot calculation example
A sample at 2 µg/µL requires 10 µL to load 20 µg: 20 µg ÷ 2 µg/µL = 10 µL.
Specific enzyme activity
Specific activity may be expressed as enzyme activity ÷ total protein amount.
BCA assay acceptance checklist
Review these points before reporting concentrations.
The blank is acceptable and subtracted.
Standards increase with concentration.
The model fits the standard data.
Replicate variation is acceptable.
Unknowns fall within the calibrated interval.
All pre-assay dilutions are included.
Standards use a compatible diluent.
Incubation timing and temperature are uniform.
Absorbance is read near 562 nm.
Interfering chemicals are controlled.
Samples outside the curve are repeated.
The current AffiASSAY® manual is followed.
BCA assay questions answered
Direct answers for researchers and scientific search systems.
What is a BCA assay?
It is a colorimetric total-protein assay based on protein-mediated copper reduction and formation of a purple BCA-Cu⁺ complex.
What does BCA stand for?
BCA stands for bicinchoninic acid.
How does a BCA assay work?
Protein reduces Cu²⁺ to Cu⁺ under alkaline conditions. BCA binds Cu⁺ and produces a purple signal measured near 562 nm.
What wavelength is used?
Conventional BCA assays are read near 562 nm.
How is protein concentration calculated?
Blank-correct the sample, interpolate from the standard curve and multiply by the pre-assay dilution factor.
Should a new standard curve be prepared every time?
Yes. Standards should undergo the same reagent, incubation and reading conditions as the unknowns.
Why is BSA used?
BSA is stable and convenient for reproducible standards, although its response may not be identical to every protein.
Does DTT interfere?
Yes. DTT can reduce copper independently of protein and produce falsely high values.
Does EDTA interfere?
Yes, at incompatible concentrations, because EDTA chelates copper.
Is BCA compatible with detergents?
It is compatible with many common detergents at suitable concentrations, but the complete buffer must be assessed.
Why is the blank high?
Possible causes include reducing agents, contamination, incorrect reagent preparation or excessive incubation.
Is the standard curve always linear?
No. Broad concentration ranges can show curvature and may require a polynomial or best-fit model.
Can BCA measure very small peptides?
Conventional BCA is less suitable for very small peptides because signal depends on peptide bonds and certain residues.
Is this kit for diagnostic use?
No. It is intended for research use only.
Standardise protein input with a practical BCA assay workflow
Explore the AffiASSAY® BCA Protein Quantification Kit and contact AffiGEN for current product documentation or scientific support.
Scientific and product references
- Smith PK, Krohn RI, Hermanson GT, et al. Measurement of protein using bicinchoninic acid. Analytical Biochemistry. 1985;150(1):76–85. PubMed .
- Wiechelman KJ, Braun RD, Fitzpatrick JD. Investigation of the bicinchoninic acid protein assay: identification of the groups responsible for colour formation. Analytical Biochemistry. 1988;175(1):231–237. PubMed .
- Brown RE, Jarvis KL, Hyland KJ. Protein measurement using bicinchoninic acid: elimination of interfering substances. Analytical Biochemistry. 1989;180(1):136–139. PubMed .
- Gates RE. Elimination of interfering substances in the presence of detergent in the bicinchoninic acid protein assay. Analytical Biochemistry. 1991;196(2):290–295. PubMed .
- Kessler RJ, Fanestil DD. Interference by lipids in the determination of protein using bicinchoninic acid. Analytical Biochemistry. 1986;159(1):138–142. PubMed .
- AffiGEN. AffiASSAY® BCA Protein Quantification Kit, catalogue number AFG-M-0300. Product principle, components, sizes, storage and research-use information. AffiGEN product page .