Input Data
Enter one value per line or separate values with commas.
Example Data Table
| Tube | Standard Concentration (µg/mL) | Absorbance at 595 nm | Blank-Corrected Absorbance |
|---|---|---|---|
| Blank | 0 | 0.020 | 0.000 |
| S1 | 100 | 0.086 | 0.066 |
| S2 | 200 | 0.149 | 0.129 |
| S3 | 400 | 0.280 | 0.260 |
| S4 | 600 | 0.405 | 0.385 |
| S5 | 800 | 0.532 | 0.512 |
| S6 | 1000 | 0.662 | 0.642 |
Formula Used
1. Blank correction
Corrected absorbance = Raw absorbance − Blank absorbance
2. Calibration line
A = mC + b
Where A is corrected absorbance, C is concentration, m is slope, and b is intercept.
3. Unknown concentration
C = (Aunknown − b) / m
4. Dilution adjustment
Final concentration = Estimated concentration × Dilution factor
5. Precision check
CV% = (Standard deviation / Mean) × 100
How to Use This Calculator
- Measure the blank, standards, and unknown sample absorbances at 595 nm.
- Enter matching standard concentrations and absorbance readings in the first two fields.
- Enter one or more unknown absorbance readings for replicate analysis.
- Provide the blank absorbance and the dilution factor used during preparation.
- Press Submit to generate the regression line, concentration estimates, and precision summary.
- Use the CSV and PDF buttons to export the displayed result section.
Assay Response Range
The Bradford assay estimates protein concentration from Coomassie dye binding measured at 595 nm. Reliable interpretation depends on standards spanning the expected sample range without forcing interpolation beyond the useful response window. A set from 0 to 1000 µg/mL provides broad coverage, but the best precision usually appears in the middle region. This calculator converts readings into concentration values through blank correction and linear regression.
Blank Correction
Blank subtraction removes signal from reagent, solvent, plate, and instrument background. If a blank of 0.020 is ignored, every point shifts upward and the regression intercept changes. That effect is small for concentrated samples, but it can bias low-concentration unknowns. Corrected absorbance makes the standard curve represent protein-dependent color development more accurately and improves comparability between runs.
Curve Quality Metrics
Slope, intercept, and R² summarize how well the standards define the calibration line. An R² near 0.99 indicates that corrected absorbance changes are closely explained by concentration. Lower values may reflect pipetting error, poorly mixed standards, contaminated wells, or use of concentrations outside the linear range. When fit quality drops, analysts should inspect standards before trusting reported unknown concentrations.
Unknown Replicate Precision
Replicate unknown readings help distinguish a true concentration result from a noisy measurement. Tight replicate clustering produces lower standard deviation and lower coefficient of variation. In many laboratory workflows, a CV under 10% is a practical target for repeatability. Higher variation may indicate bubbles, timing differences, incomplete mixing, or sample heterogeneity that should be reviewed before final reporting.
Dilution Factor Impact
Many samples must be diluted so their absorbance falls inside the calibration range. The calculator reports both estimated concentration in the assay mixture and adjusted concentration in the original sample. For example, an interpolated value of 32.1 µg/mL with a dilution factor of 10 becomes 321 µg/mL. Showing both values reduces transcription mistakes and improves traceability in worksheets and exported records.
Workflow Value
Combining standards, replicates, statistics, and a curve on one page supports review during teaching, screening, and bench work. Analysts can enter data, evaluate fit quality, confirm replicate precision, and export results without recalculating values manually. This structure turns absorbance readings into a concentration summary that is easier to archive and communicate across laboratory teams.
FAQs
1. What does this calculator estimate?
It estimates protein concentration from Bradford assay absorbance data using blank correction, linear regression from standards, and dilution factor adjustment for the original sample.
2. Why is blank correction necessary?
Blank correction removes background signal from reagent and instrument effects. Without it, calculated concentrations can be biased upward, especially for low-concentration unknowns.
3. What does a low R² value mean?
A low R² suggests the standards do not fit the line well. Common causes include pipetting error, inconsistent mixing, outlier standards, or use outside the linear range.
4. How should I enter replicate readings?
Enter each unknown absorbance as a separate line or comma-separated value. The calculator will compute replicate concentration, mean, standard deviation, and CV automatically.
5. When should I apply a dilution factor?
Apply a dilution factor when the sample was diluted before measurement. The calculator multiplies the interpolated assay concentration by that factor to recover the original concentration.
6. Can I export the calculated results?
Yes. The result section can be exported as CSV for tabular records and as PDF for sharing, printing, or adding to laboratory documentation.