Protein Concentration Calculator
Calculate protein concentration from A280 absorbance using extinction coefficient. Uses Beer-Lambert law: c = A280 / (epsilon x path length). Supports both absorbance and Lowry/Bradford indirect methods.
🧪 Biochemistry📐 c = A280 / (epsilon x l)🧬 Biology
Absorbance at 280nm (A280)
Extinction coefficient (M-1cm-1)
Molecular weight (Da)
Path length (cm)
Please enter valid values.
Formula & Reference
| Variable | Symbol | Formula | Units |
|---|---|---|---|
| Protein Concentration Calculator | — | c = A280 / (epsilon x l) | mg/mL |
Step-by-Step Examples
Example 1
BSA Standard
BSA A280=0.65, epsilon=43824 M-1cm-1, MW=66430 Da.
- c(M) = 0.65 / 43824 = 14.83 uM
- c(mg/mL) = 14.83e-6 x 66430 = 0.985 mg/mL
- Or use A280 x 1.45 as rough estimate for BSA
- Final: ~1 mg/mL BSA
✓ c = 0.985 mg/mL BSA
Example 2
GFP Protein
GFP A280=1.25, epsilon=21890 M-1cm-1, MW=26900 Da.
- c(M) = 1.25/21890 = 57.1 uM
- c(mg/mL) = 57.1e-6 x 26900 = 1.536 mg/mL
- GFP also absorbs at 488nm (check A488 for functional protein)
✓ c = 1.54 mg/mL GFP
Example 3
Unknown Protein
Purified protein with unknown epsilon: A280=0.4, MW=50000 Da. Use A280 x 1.0 as rough estimate.
- Rough estimate: c ~ 0.4 mg/mL
- For accuracy: determine epsilon from sequence (ExPASy ProtParam)
- Then use Beer-Lambert for precise concentration
✓ Rough: ~0.4 mg/mL (use actual epsilon for accuracy
Real-World Applications
Enzyme Assays
Enzyme units (U/mg) require accurate protein concentration to calculate specific activity.
Therapeutic Proteins
Precise concentration of antibodies and recombinant proteins critical for dosing.
SDS-PAGE Loading
Equal loading of protein gels requires accurate quantification.
Biophysical Studies
ITC, SPR, and NMR require known protein concentrations for accurate thermodynamic measurements.
Common Mistakes to Avoid
⚠️
Using wrong extinction coefficient
Epsilon depends on the number of Trp and Tyr residues. Use ExPASy ProtParam to calculate sequence-specific epsilon, not a generic value.
⚠️
Ignoring light scattering
Aggregated protein scatters light at 280nm, overestimating concentration. Check for aggregation by DLS or SEC first.
⚠️
Forgetting to subtract buffer background
A280 of buffer must be subtracted from sample A280 before calculating concentration.
Frequently Asked Questions
Where do I find protein extinction coefficient? ▾
Use ExPASy ProtParam (web.expasy.org/protparam). Paste sequence and get epsilon based on Trp (5500 M-1cm-1) and Tyr (1490 M-1cm-1) content.
What if my protein has no Trp or Tyr? ▾
A280 method won't work. Use Bradford, BCA, or amino acid analysis instead. Or measure at 205nm (peptide bonds absorb there).
What is the BCA assay? ▾
Bicinchoninic acid assay — colorimetric method at 562nm. More sensitive than Bradford, works with detergents, less susceptible to protein type differences.
What is Bradford assay? ▾
Coomassie G-250 dye binding assay at 595nm. Simple, fast, but varies with protein type and requires standard curve.
How does molecular weight affect units? ▾
Molarity (M) = A280/epsilon. mg/mL = molarity x MW (g/mol). Choosing units depends on experiment — molarity for binding studies, mg/mL for biological assays.