The BCA (Bicinchoninic Acid) protein assay is a straightforward procedure that quantifies protein concentration by forming a colorimetric complex between bicinchoninic acid and reduced copper ions (Cu⁺) in the presence of proteins. Below is a general protocol for performing a BCA protein assay:
Materials Needed
BCA Protein Assay Kit (includes Reagent A and Reagent B)
Protein standards (e.g., Bovine Serum Albumin, BSA)
Protein samples
Microplate reader or spectrophotometer (capable of reading absorbance at 562 nm)
96-well plate or cuvettes (depending on equipment)
Pipettes and tips
Incubator or heating block (optional)
Preparation
- Prepare protein standards:
Dilute a stock solution of BSA to create a series of known concentrations (e.g., 25, 125, 250, 500, 1000, and 2000 µg/mL). This will be used to generate a standard curve.
For each standard concentration, use 50 µL in a microplate well (or more if using cuvettes).
- Prepare working reagent:
Mix 50 parts of Reagent A (BCA reagent) with 1 part of Reagent B (Cu²⁺ sulfate solution).
For example, mix 50 mL of Reagent A with 1 mL of Reagent B for 51 mL of working reagent. The amount needed depends on the number of wells or cuvettes you plan to process.
Protocol Steps
- Sample Preparation:
Prepare your samples in the appropriate buffer. Ensure that any interfering substances (e.g., high concentrations of reducing agents) are minimized, as they could affect the assay.
Add 50 µL of each sample (or a suitable volume for your equipment) to wells of a 96-well plate or to a cuvette.
- Add Working Reagent:
To each well (or cuvette), add 200 µL of the BCA working reagent.
Ensure the reagent and sample are well mixed. Gently tap or shake the plate to mix if necessary.
- Incubation:
Incubate the plate or cuvettes at 37°C for 30 minutes (standard protocol) or at room temperature for about 2 hours. The temperature can slightly affect the color development, but 37°C generally provides faster results.
Cover the plate during incubation to prevent evaporation.
- Measure Absorbance:
After incubation, measure the absorbance of the samples at 562 nm using a microplate reader or a spectrophotometer.
Ensure that the blank (typically buffer without protein) is included for background correction.
- Data Analysis:
Subtract the absorbance of the blank from all sample and standard readings.
Generate a standard curve by plotting the absorbance of the known protein standards against their concentrations.
Use the standard curve to determine the protein concentration of your unknown samples by interpolating their absorbance values.
Tips
Standard curve: Always include a set of known protein standards to ensure accurate quantification. BSA is a common choice, but using a standard protein similar to your sample protein can improve accuracy.
Sample handling: If your sample contains interfering substances, such as high concentrations of detergents or reducing agents, consider diluting the sample or using a compatible assay buffer.
Incubation: The reaction is linear within a certain range, but prolonged incubation can lead to overestimation of protein concentration, so stick to the recommended incubation time.
Example BCA Assay Plate Setup (96-well plate):
| Well | Content |
| A1–A6 | Blank (buffer only) |
| B1–B6 | Protein standards (various concentrations) |
| C1–C6 | Sample 1 (replicates) |
| D1–D6 | Sample 2 (replicates) |
This protocol will give reliable, reproducible results when quantifying protein concentrations in various samples.