Description
BCA protein quantification kit
GenieBlot BCA Protein Quantification Kit is a colorimetric total-protein assay based on the bicinchoninic acid (BCA) reaction, one of the most sensitive methods for measuring protein concentration. It offers a broad working range (~20–2000 μg/ml), high detergent tolerance and compatibility with both microplate and cuvette formats.
Under alkaline conditions, protein reduces Cu2+ to Cu+, which then reacts with BCA to form a water-soluble purple complex with strong absorbance at 562 nm. Absorbance is linear with protein concentration over a wide range, so sample concentrations are read directly from a BSA standard curve. Common concentrations of detergents such as SDS, Triton X-100 and Tween-20 do not affect results, making the kit suitable for proteins extracted with most common lysis buffers. Chelating agents (EDTA, EGTA), reducing agents (DTT, mercaptoethanol) and lipids can interfere. For research use only.
Key Features
| Simple operation | Mix the working solution, add samples, incubate 20–30 min at 37°C and read at 562 nm. | |
| High sensitivity | One of the most sensitive colorimetric methods for total protein. | |
| Broad compatibility | Tolerates common ionic and non-ionic detergents, including 3% SDS and 5% Triton X-100. | |
| Linear standard curve | Absorbance is linear with protein concentration over a wide range. |
Specifications
| Method | Bicinchoninic acid (BCA) colorimetric assay |
| Detection | Absorbance at 562 nm (microplate reader or spectrophotometer) |
| Working range | ~20–2000 μg/ml |
| Sample volume | 20 μl per well (96-well microplate format) |
| Assay time | 20–30 min incubation at 37°C |
| Samples | Protein samples extracted with common lysis buffers |
| Shelf life | 2 years |
Components
| Components | 250 rxns | 500 rxns |
| BSA Standard (1 mg/ml) | 2 × 1 ml | 4 × 1 ml |
| BCA Reagent A | 50 ml | 100 ml |
| BCA Reagent B | 1 ml | 2 × 1 ml |
Storage
Store at 15 ~ 25°C.
96-well microplate assay
- Prepare BCA working solution
- Mix BCA Reagent A and BCA Reagent B at a volume ratio of 50:1, enough for all samples. Use fresh tips for each reagent and use the working solution immediately.
- Set up the standard curve
- Add reagents to a microplate as follows:
| Well no. | 0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| Protein standard (μl) | 0 | 1 | 2 | 4 | 8 | 12 | 16 | 20 |
| ddH2O (μl) | 20 | 19 | 18 | 16 | 12 | 8 | 4 | 0 |
| BCA working solution (μl) | 200 | 200 | 200 | 200 | 200 | 200 | 200 | 200 |
| Protein content (μg) | 0 | 1 | 2 | 4 | 8 | 12 | 16 | 20 |
- Prepare samples
- Dilute each protein sample to a suitable concentration with ddH2O. Add 20 μl of sample to 200 μl of BCA working solution.
- Incubate
- Mix by shaking and incubate at 37°C for 20–30 min.
- Measure absorbance
- Read absorbance at 562 nm on a microplate reader. The well without BSA is the blank.
- Calculate protein concentration
- Plot protein content (μg, x-axis) against absorbance (y-axis).
- Read the protein content of each sample from the standard curve, divide by the 20 μl sample volume and multiply by the dilution factor.
Cuvette assay
Follow the same procedure, scaling up all volumes in proportion to the cuvette size.
Compatible substance concentrations
Upper concentration limits of substances that do not interfere with the assay:
| Substance | Compatible concentration |
| Glucose | 10 mM |
| Octyl glycoside | 5.0% |
| Sodium acetate, pH 5.5 | 200 mM |
| Sucrose | 40% |
| Ammonium sulfate | 1.5 M |
| Brij-35 | 5.0% |
| CHAPS | 5.0% |
| DTT | 0.5 mM |
| EDTA | 15 mM |
| Emulgen | 1.0% |
| Glycine, pH 2.8 | 100 mM |
| Guanidine·HCl | 4.0 M |
| HEPES | 100 mM |
| Tween-20 | 5.0% |
| NaOH | 0.1 M |
| NP-40 | 5.0% |
| SDS | 3.0% |
| Sodium chloride | 1.0 M |
| Triton X-100 | 5.0% |
| Urea | 3.0 M |
Q1: Which substances interfere with the assay?
- Common detergents do not affect results, but chelating agents (EDTA, EGTA), reducing agents (DTT, mercaptoethanol) and lipids do. Tolerated limits include 3.0% SDS, 5.0% Triton X-100, 5.0% Tween-20, 15 mM EDTA, 0.5 mM DTT and 0.07% β-mercaptoethanol; see the compatibility table in the protocol for more.
Q2: Can I use a spectrophotometer instead of a microplate reader?
- Yes. Scale up the working solution so it is at least the cuvette's minimum volume, and scale samples and standards proportionally. Fewer samples can be measured this way. Plastic cuvettes are recommended.
Q3: The BCA reagent has crystallised. Can I still use it?
- Yes. Warm it at 37°C until the crystals dissolve completely; performance is not affected.
Q4: What if my lysis buffer gives a high background?
- A Bradford protein assay is recommended for sample diluents or lysis buffers with high background values.
