Principles of Protein Concentration Determination
Quantifying protein concentration is essential in biochemistry, molecular biology, structural biology, and bioprocessing. The protein concentration calculator provides rapid estimation of concentration using A280 absorbance or assay-derived signals, enabling accurate quantification for purification, enzyme assays, and downstream experimental workflows.
Absorbance Measurements and the Beer–Lambert Law
Direct UV absorbance at 280 nm is a widely used method for protein quantification. This measurement follows the Beer–Lambert Law:
A = ε × c × l
where A is absorbance, ε is the extinction coefficient, c is concentration, and l is the optical path length. This calculator uses this relationship to determine mass concentration from the measured absorbance.
Using Calibration-Based Assays for Concentration
Colorimetric assays such as Bradford, BCA, and Lowry generate absorbance values proportional to protein content. When supplied with a calibration factor or standard curve, this calculator can compute protein concentration using measured optical density values.
Converting Between Mass and Molar Concentration
If molecular weight is provided, the calculator can convert mg/mL to molar concentration:
- Molarity (M): concentration ÷ MW
- µM: molarity × 10⁶
This is especially useful for enzyme kinetics, binding studies, and stoichiometry calculations.
Applications Across Biochemical Workflows
- Protein purification and chromatography analysis
- Enzyme assays and kinetic studies
- Sample normalization prior to SDS-PAGE or Western blotting
- Bioreactor and fermentation monitoring
- Structural biology and crystallography preparations
Best Practices for Reliable Protein Quantification
- Use clean cuvettes or validated microplate paths.
- Account for buffer absorbance using a blank measurement.
- Ensure extinction coefficients match the protein sequence.
- Avoid measurements outside the linear absorbance range.
Accurate quantification using a protein concentration calculator supports reproducible experimental workflows and precise biochemical measurements.
FAQ
Protein Quantification Questions
Key concepts for absorbance-based and assay-based protein measurement.
It uses Beer–Lambert Law (A = ε × c × l) or assay calibration factors to calculate protein concentration from absorbance or mass-based inputs.
It relates absorbance to concentration using A = ε × c × l, where ε is the extinction coefficient, c is concentration, and l is path length.
Yes. The calculator supports A280 measurements when the extinction coefficient of the protein is known.
Yes. Enter the assay slope or calibration constant to estimate protein concentration from colorimetric absorbance values.
The calculator supports mg/mL, µg/mL, molarity, and any absorbance-based concentration depending on the input values.
Use the protein’s known extinction coefficient at 280 nm, which is typically provided by sequence-based calculators or published data.
Yes. Enter molecular weight, and the calculator can interconvert mg/mL and molar concentration.
Standard cuvettes have a 1 cm path length, but microplate readers may use shorter or variable path lengths.
Yes. As long as absorbance or assay signals are reliable, the calculator can estimate concentration for both purified and crude samples.
Yes. Light scattering from particulates can artificially increase absorbance, leading to inaccurate concentration estimates.