Protein Concentration Calculator

About the Protein Concentration Calculator

Protein concentration determination is one of the most frequent assays in biochemistry and molecular biology laboratories. From cell lysate quantification before Western blots to enzyme kinetics substrate preparation, virtually every protein experiment begins with knowing "how much protein do I have?"

Three dominant methods cover most laboratory needs. **UV absorbance at 280 nm (A280)** exploits the aromatic amino acids tryptophan and tyrosine to directly measure protein without reagents — fast and non-destructive, but requires knowing the extinction coefficient and a pure sample. **The Bradford assay** uses Coomassie Brilliant Blue G-250 dye binding, shifting absorbance from 465 to 595 nm proportionally to protein concentration — the workhorse of most laboratories. **The BCA assay** (bicinchoninic acid) reduces Cu²⁺ to Cu⁺ in the presence of protein, producing a purple complex measured at 562 nm — more tolerant of detergents than Bradford.

This calculator handles all three methods: direct A280 calculation with custom extinction coefficients, standard curve fitting from Bradford/BCA absorbance data, and unit conversions between mg/mL, µM, and µg/µL. It also calculates dilutions to target concentrations and total protein yields from cell lysates.

When This Page Helps

Accurate protein quantification is the starting point for equal loading in SDS-PAGE/Western blots, standardized enzyme activity measurements, protein crystallography trials, and virtually every quantitative biochemistry experiment.

How to Use the Inputs

1. Select quantification method: A280, Bradford, or BCA
2. For A280: enter absorbance, extinction coefficient, and path length
3. For Bradford/BCA: enter your BSA standard data points
4. Enter your unknown sample absorbance reading
5. Review concentration in mg/mL and molar units
6. Use the dilution calculator for target concentration preparation
7. Check total protein yield from your sample volume

Example Calculation

Tips & Best Practices

• Always run a fresh standard curve with each batch of Bradford or BCA reagent
• Blank A280 readings with your buffer — imidazole and DTT absorb at 280 nm
• For Bradford, add sample to reagent (not the reverse) to avoid dye precipitation
• BCA assay is more consistent but slower (30 min at 37°C vs 5 min for Bradford)
• Store BSA standards as 2 mg/mL stock aliquots at -20°C — avoid repeated freeze-thaw
• Use glass or low-bind plastic for dilute protein samples (<10 µg/mL) to prevent adsorption losses

Standard Curve Best Practices

A reliable standard curve requires at least 5-7 data points spanning the linear range, plus a blank (zero protein). Prepare standards by serial dilution from a single concentrated BSA stock (2 mg/mL, weighed and dissolved fresh or purchased certified). Read all standards and unknowns in the same plate/batch. For Bradford: incubate exactly 5 minutes before reading — color continues to develop. For BCA: incubate 30 min at 37°C (standard) or 2 hours at room temp (enhanced protocol). Report R² of the linear fit — should be >0.99 for valid quantification.

Protein-Specific Extinction Coefficients

Every protein has a unique ε₂₈₀ based on its aromatic amino acid content. Common reference proteins: **BSA**: 43,824 M⁻¹cm⁻¹ (ε₀.₁% = 0.667). **IgG (human)**: ~210,000 M⁻¹cm⁻¹ (ε₀.₁% = 1.35). **Lysozyme**: 37,970 M⁻¹cm⁻¹ (ε₀.₁% = 2.65). Use ExPASy ProtParam to calculate ε₂₈₀ from your protein's amino acid sequence — it's instantaneous and removes any guesswork.

Detergent Compatibility Guide

**Bradford (Coomassie)**: Incompatible with SDS (>0.1%), Triton X-100 (>0.1%), NP-40. Compatible with: urea (up to 4M), NaCl, EDTA, Tris. **BCA**: Compatible with SDS (up to 5%), Triton (up to 5%), NP-40 (up to 5%). Incompatible with reducing agents (DTT, β-ME) in standard protocol. **A280**: Compatible with most buffers. Incompatible with nucleic acids (inflate reading) and DTT/imidazole (absorb at 280 nm). Choose your assay based on what's in your sample buffer.

Frequently Asked Questions

• Which assay should I use?

  A280 for pure proteins when you know the extinction coefficient. Bradford for most routine lab work (fast, cheap, 5-minute protocol). BCA when your samples contain detergents (SDS, Triton X-100) — Bradford is incompatible with most detergents. For ELISA plates or high-throughput: micro BCA or Bradford microplate formats.

• What is the extinction coefficient?

  The molar extinction coefficient (ε) describes how strongly a protein absorbs UV light at 280 nm. It depends on the number of Trp (ε₂₈₀ = 5,500 M⁻¹cm⁻¹), Tyr (1,490), and Cys-Cys (125) residues. ProtParam (ExPASy) calculates it from sequence. The mass extinction coefficient (E₁%₁cm or ε₀.₁%) is ε / MW × 10, giving absorbance of a 1 mg/mL solution.

• Why does Bradford reagent turn blue?

  Coomassie G-250 exists in three forms: cationic (red, 465 nm), neutral (green, 650 nm), and anionic (blue, 595 nm). In acidic conditions, the dye is predominantly red. When it binds protein (via electrostatic and hydrophobic interactions with basic and aromatic residues), it shifts to the blue anionic form. The increase in A595 is proportional to protein concentration.

• What is the linear range of Bradford and BCA?

  Standard Bradford: 100-1,500 µg/mL (linear). Micro Bradford: 1-25 µg/mL. Standard BCA: 20-2,000 µg/mL. Micro BCA: 0.5-20 µg/mL. Always verify linearity with your specific standards — lot-to-lot variation in reagents can shift the range.

• Can I use A280 for crude lysates?

  Not accurately. Nucleic acids absorb strongly at 260 nm with significant carryover to 280 nm, inflating the apparent protein concentration. The Warburg-Christian correction (protein (mg/mL) = 1.55×A280 - 0.76×A260) partially compensates but is imprecise. Use Bradford or BCA for crude lysates.

• How accurate is the BSA standard curve for my protein?

  Bradford and BCA both show protein-to-protein variation. The response per mg is not identical for BSA and your target protein because dye binding depends on amino acid composition. BSA is the conventional reference standard, so concentrations are reported as "BSA equivalents." For absolute quantification, use amino acid analysis.