BCA protein assay
Proteins reduce copper ions and BCA turns purple in proportion. A standard curve from known concentrations (BSA) converts each sample's absorbance into a concentration.
Key points
- Dilute samples so they land inside (ideally the middle of) the standard range
- Reducing agents (DTT, β-ME) and chelators (EDTA) inflate the reading
- Standards and samples must share the same buffer and incubation time
Materials
- BCA reagents A and B
- BSA standard (2 mg/mL)
- The same buffer as the samples (for standards and blank)
- Clear 96-well plate
- 37 °C incubator
- Plate reader at 562 nm
Steps
- 💡 If unsure, run two dilutions.
Calculations
Working reagent (200 µL per well)
🔬 Variable explorer — what changes if…?
The skeleton stays the same, but concentrations, times and reagents change between experiments. Click an item to see what it affects and compare two conditions (A and B) side by side.
Sample dilution
What it isHow much to dilute the sample. Readings are only reliable inside the standard range.
Less dilutionToo little dilution: above the top standard, outside the curve (unreliable, underestimated).
More dilutionToo much dilution: near the bottom, where small errors are magnified.
Typical choiceAround the middle of the curve (inside the green band above).
Incubation temperature · time
What it isThe colour keeps developing, so temperature and time change sensitivity and range.
Lower · lessRoom temperature or short: lower sensitivity for dilute samples.
Higher · more60 °C or long: more sensitive, but top standards saturate and the range shrinks.
Typical choice37 °C for 30 min — always identical for standards and samples.
Buffer components (interference)
What it isLysis buffer components can interfere with colour development.
Typical choicePick the method that suits your buffer, and make blank and standards in that buffer.
Standard protein
What it isValues against BSA are concentrations relative to BSA. For antibody samples an IgG standard is sometimes used.
Typical choiceUsing the same standard throughout keeps samples comparable.
Troubleshooting
| Problem | Common cause | Fix |
|---|---|---|
| Poor standard curve (low R²) | Pipetting error, bubbles, wrong standard dilutions | Remake standards, remove bubbles, use reverse pipetting |
| Samples above the top standard | Not diluted enough | Dilute further and re-read |
| Blank is also strongly purple | Reducing agents or chelators react | Check the buffer, dilute out the interferent, or use a compatible kit |
| Large differences between replicates | Poor mixing, edge evaporation | Shake to mix and keep covered |
⚠ Safety Reagent B contains copper sulfate — avoid contact with eyes and skin.
This page summarises widely used general conditions. Reagent, kit and antibody manuals and your institution's safety rules take priority. Simulators marked "concept" are simplified models that show trends.
Analyse the results with free tools:
Standard curve calculator (BCA, ELISA) → Reagent & dilution calculator →