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Protein Concentration Calculator

Ready to calculate
c = A / (ε × l).
14 protein presets.
5 concentration units output.
100% Free.
No Data Stored.

How it Works

01Pick a Protein (or Custom)

14 presets with auto-filled ε and MW (BSA, IgG, lysozyme, insulin, PE, APC, RNase A, etc.) or enter your own.

02Measure Absorbance

UV-Vis spectrophotometer at λmax (typically 280 nm). Best quantitation: A = 0.1-1.5; outside this range, dilute or concentrate.

03Apply c = A / (ε × l)

Beer-Lambert law gives molar concentration in cuvette. Multiply by dilution factor for original sample concentration.

04Get Concentration in 5 Units

Output: M, mM, µM, mg/mL, µg/mL — pick whichever your downstream protocol uses.

What is a Protein Concentration Calculator?

Protein concentration is the most-measured biochemical quantity in research and diagnostic laboratories — every Western blot, every ELISA, every enzyme kinetics assay, every protein-purification step starts with knowing how much protein is in solution. Our Protein Concentration Calculator implements the standard Beer-Lambert UV-Vis spectrophotometric quantitation: c = A / (ε × l), where A is measured absorbance, ε is the molar extinction coefficient (M⁻¹·cm⁻¹), and l is the cuvette pathlength (cm). For aromatic-residue-containing proteins, the standard wavelength is 280 nm (sum of Trp, Tyr, and disulfide-bridged cystine absorbance). For special chromophores like phycoerythrin (PE, λmax 565 nm) and allophycocyanin (APC, λmax 650 nm), the calculator allows wavelength specification.

The calculator includes 14 protein presets with auto-filled extinction coefficients and molecular weights — saving you the lookup step for the most common research proteins: IgG (210,000 / 150,000), BSA (43,824 / 66,430), lysozyme (38,000 / 14,300), insulin (5,840 / 5,808), RNase A (9,800 / 13,700), streptavidin (167,760 / 53,000 tetramer), fluorescent labels phycoerythrin (1.96 × 10⁶ / 240,000) and allophycocyanin (700,000 / 105,000), individual aromatic amino acids (Trp 5,500; Tyr 1,490; Phe 200), and ATP / cysteine / aldose for special applications. Custom protein mode accepts any user-supplied ε and MW for unknowns or engineered constructs.

Multi-unit input: pathlength in cm / mm / m (standard quartz cuvette 1 cm; NanoDrop 0.1 cm). Dilution-factor field corrects automatically — enter the dilution applied before measurement (e.g. 10× for 1:10 dilution); the calculator multiplies the in-cuvette concentration by the dilution factor to give the original-sample concentration. Output: protein concentration in 5 unit systems simultaneously (M, mM, µM, mg/mL, µg/mL) with smart warnings for absorbance outside the linear quantitation range (0.1-1.5), exceptional extinction coefficients, and signal-to-noise concerns. Designed for biochemistry / molecular-biology coursework, protein-purification labs (FPLC fractions), antibody quantitation for ELISA / Western standards, fluorescent-label conjugation calculations, and any researcher needing a fast Beer-Lambert calculation — runs entirely in your browser, no account, no data stored.

Pro Tip: Pair this with our Molarity Calculator for solution preparation, our Dilution Factor Calculator for pre-measurement sample dilution, our Mole Calculator for stoichiometry, or our Molality Calculator for colligative-property work.

How to Use the Protein Concentration Calculator?

Pick a Protein Preset (or Custom): 14 common research proteins with auto-filled ε and MW — IgG, BSA, lysozyme, insulin, RNase A, streptavidin, PE, APC, individual amino acids. For unknowns or engineered proteins, pick "Custom" and enter ε and MW manually. For unknown proteins, compute ε from amino-acid sequence using ExPASy ProtParam (Edelhoch / Pace formula): ε(280 nm) = 5500 × n_Trp + 1490 × n_Tyr + 125 × n_cystine.
Verify or Override ε and MW: the preset values are typical published values. Check supplier datasheets / Certificates of Analysis for lot-specific values; engineered proteins (His-tags, GFP fusions, mutations) may have different ε and MW from wild-type.
Measure Absorbance on a UV-Vis Spectrophotometer: at the protein's λmax (280 nm for most proteins; 565 nm for PE; 650 nm for APC). Best practice: blank against the same buffer used to dissolve the protein (matrix matching); take 3 readings and average; verify A280 peak shape (a clean, narrow peak indicates pure protein; broad or shoulder peaks suggest aggregation, contamination, or denaturation).
Keep Absorbance in Linear Range (A = 0.1-1.5): below 0.1, signal-to-noise becomes poor; above 1.5, the spectrophotometer enters non-linearity (stray light, detector saturation). If A is too low: concentrate the sample (vacuum centrifuge, ultrafiltration); use longer pathlength (1 cm vs 1 mm); or use a more sensitive method (Bradford, BCA). If A is too high: dilute the sample 5× or 10× and re-measure (record dilution factor for the calculator).
Enter Pathlength: standard quartz cuvette = 1.0 cm; NanoDrop microvolume = 0.1 cm (=1 mm); UV-cuvette options 1, 2, 5, 10 mm. The calculator accepts cm / mm / m and converts internally.
Apply Dilution Factor (if applicable): enter the factor by which you diluted the sample BEFORE measurement. E.g. if you took 10 µL of stock + 90 µL of buffer (1:10 dilution), enter DF = 10. The calculator multiplies the in-cuvette result by 10 to give the original sample concentration. Default is 1 (no dilution).
Apply Beer-Lambert: c = A / (ε × l): the calculator returns concentration in M (the SI base) and converts to mM, µM, mg/mL, and µg/mL automatically. Mass concentration = molar concentration × MW.
Check the Calculation Breakdown: verify the input values, ε × l product, in-cuvette concentration before dilution correction, and the final original-sample concentration. The breakdown panel makes it easy to spot transcription errors or unit-conversion mistakes.

How is protein concentration calculated?

Protein concentration via UV-Vis is one of the cleanest applications of the Beer-Lambert law — derived from molecular absorption physics, accurate to ~5% with care, and the workhorse of every biochemistry lab worldwide. The math is simple; the experimental craft (cuvette cleanliness, sample preparation, blank choice) determines the precision.

References: Beer (1852) Ann. Phys. Chem. 86 78; Lambert (1760) Photometria; Edelhoch (1967) Biochemistry 6 1948; Pace, Vajdos, Fee, Grimsley & Gray (1995) Protein Sci. 4 2411 (extinction coefficient calculation).

Beer-Lambert Law

A = ε × c × l

Solving for concentration: c (mol/L) = A / (ε × l), where A is absorbance (dimensionless, log₁₀ of incident over transmitted intensity), ε is molar extinction coefficient (M⁻¹·cm⁻¹), and l is pathlength (cm).

Mass-Concentration Conversion

c (mg/mL) = c (mol/L) × MW (g/mol)

Algebraic check: (mol/L) × (g/mol) = g/L = mg/mL (numerically equal in dilute aqueous solutions).

Dilution-Factor Correction

If sample was diluted by factor DF before measurement: c_original = c_cuvette × DF. The calculator handles this automatically.

Example: 1:10 dilution (10 µL sample + 90 µL buffer) gives DF = 10. If A = 0.5, ε = 100,000, l = 1 cm: c_cuvette = 0.5 / 100,000 = 5 µM; c_original = 5 × 10 = 50 µM.

Worked Example — IgG Antibody Quantitation

Measure 1.4 mg/mL IgG stock; 1:10 dilution gives A280 = 0.196 in 1 cm cuvette.

  • ε(IgG at 280 nm) = 210,000 M⁻¹·cm⁻¹; MW = 150,000 g/mol.
  • c_cuvette = 0.196 / (210,000 × 1) = 9.33 × 10⁻⁷ M = 0.933 µM.
  • c_original = 0.933 × 10 = 9.33 µM = 1.40 mg/mL ✓ (matches expected 1.4 mg/mL stock).

Worked Example — BSA Standard

Bovine serum albumin (BSA) standard solution. A280 = 0.660 in 1 cm cuvette, no dilution.

  • ε(BSA at 280 nm) = 43,824 M⁻¹·cm⁻¹; MW = 66,430 g/mol.
  • c = 0.660 / (43,824 × 1) = 1.506 × 10⁻⁵ M = 15.06 µM.
  • c (mg/mL) = 15.06 × 10⁻⁶ × 66,430 = 1.000 mg/mL.
  • This is the canonical BSA standard concentration: 1 mg/mL gives A280 = 0.66 (the famous "BSA = 0.66 per mg/mL" rule of thumb).

Reference Extinction Coefficients (M⁻¹·cm⁻¹) at 280 nm Unless Noted

  • BSA (Bovine Serum Albumin): 43,824 (MW 66,430).
  • IgG (Immunoglobulin G): 210,000 (MW ~150,000).
  • Lysozyme: 38,000 (MW 14,300).
  • Insulin: 5,840 (MW 5,808).
  • RNase A: 9,800 (MW 13,700).
  • Streptavidin (tetramer): 167,760 (MW 53,000).
  • Phycoerythrin (PE) at 565 nm: 1,960,000 (MW 240,000) — highest natural ε.
  • Allophycocyanin (APC) at 650 nm: 700,000 (MW 105,000).
  • GFP at 488 nm: 56,000 (MW 27,000).
  • mCherry at 587 nm: 72,000 (MW 27,000).
  • Tryptophan (Trp): 5,500 (MW 204).
  • Tyrosine (Tyr): 1,490 (MW 181).
  • Cystine (disulfide): 125 (per disulfide bond).
  • ATP at 259 nm: 15,400 (MW 507).

Extinction Coefficient from Sequence (Edelhoch / Pace Formula)

For unknown or engineered proteins, compute ε(280 nm) from amino-acid composition:

ε(280) = 5500 × n_Trp + 1490 × n_Tyr + 125 × n_cystine

Where n_cystine is the number of disulfide bridges (= half the number of cystine residues forming disulfides; reduced cysteine contributes negligibly). Implemented in ExPASy ProtParam (web.expasy.org/protparam/) — paste FASTA sequence to get ε and MW automatically.

Real-World Example

Worked Example — Quantify Antibody Stock for ELISA Standard

Scenario: A research lab purified an IgG monoclonal antibody by Protein A affinity chromatography. The eluted fraction is concentrated by ultrafiltration; final volume ~500 µL. Goal: quantify concentration accurately for ELISA standard preparation (need 1.0 mg/mL working solution).

Step 1 — Dilute Sample for Linear-Range Measurement.

  • Stock estimated > 5 mg/mL (concentrate of 0.5 mL from large pool).
  • At 5 mg/mL × 1 cm × 210,000 / (1 mg/mL × 0.66 BSA-equivalent) ≈ A280 = 7.0 — way above linear range (1.5 max).
  • Dilute 1:20 (5 µL stock + 95 µL buffer) → expected A280 ≈ 0.35 (mid-range).

Step 2 — Measure Diluted Sample.

  • Blank against 100 µL of identical buffer.
  • Take three A280 readings; average = 0.392.
  • Verify clean peak shape on full UV-Vis scan (220-340 nm); should see Trp/Tyr peak at 280 with clean baseline at 320 nm.
  • Calculate A260/A280 ratio = 0.215/0.392 = 0.55 → typical for pure protein (DNA/RNA contamination would push this above 1.0).

Step 3 — Apply Beer-Lambert + Dilution Correction.

  • ε(IgG at 280 nm) = 210,000 M⁻¹·cm⁻¹; MW = 150,000 g/mol.
  • l = 1 cm (standard cuvette).
  • c_cuvette = 0.392 / (210,000 × 1) = 1.867 × 10⁻⁶ M = 1.867 µM.
  • c_original = 1.867 × 20 = 37.33 µM (DF = 20).
  • c (mg/mL) = 37.33 × 10⁻⁶ × 150,000 = 5.60 mg/mL.

Step 4 — Prepare 1.0 mg/mL Working Standard.

  • Dilution factor needed: 5.60 / 1.0 = 5.60×.
  • For 1 mL of 1 mg/mL: take 1000 / 5.60 = 178.6 µL of stock + buffer to total 1.0 mL.
  • Verify by re-measuring at 1.0 mg/mL: expected A280 = 1.40 — slightly above linear range (1.5 limit), so dilute 1:2 for verification (expected A = 0.7 in cuvette).

Step 5 — Document and Store.

  • Label: "IgG mAb [name], 1.00 mg/mL, [date], [initials]; verified by A280, ε = 210,000".
  • Store at 4 °C for short-term (1-4 weeks) or aliquot and freeze at −80 °C for long-term.
  • Add 0.02% sodium azide as preservative for 4 °C storage; verify no interference with downstream ELISA before adding.
  • Re-quantify before each major experiment if > 4 weeks old; antibodies can lose activity (and apparent A280 stays the same as the protein concentration, but binding capacity may have dropped).

Who Should Use the Protein Concentration Calculator?

1
Quantify protein in eluted fractions to identify peak fractions for pooling. Combine with SDS-PAGE for purity assessment. Standard workflow in any protein-purification project.
2
Standardize IgG concentration before ELISA or Western blot. ε(IgG) = 210,000 M⁻¹·cm⁻¹ at 280 nm; 1 mg/mL gives A280 ≈ 1.4. Critical for reproducible immunoassays.
3
Compute molar enzyme concentration for Michaelis-Menten and turnover-number (kcat) calculations. ε from sequence via ExPASy ProtParam for engineered constructs.
4
Compute degree of labeling (DoL) for fluorescent-dye conjugates. Standard formula: DoL = (A_dye × ε_protein) / ((A_280 − CF × A_dye) × ε_dye), where CF is correction factor for dye absorbance at 280 nm.
5
Quantify protein yield from E. coli, mammalian, or yeast expression systems. Compare against expected yield (typically 0.5-50 mg/L for bacterial expression).
6
Quantify therapeutic-protein candidates (mAbs, fusion proteins, vaccines) for dose preparation. USP / EP titrations use UV-Vis quantitation as the primary release-test method.
7
Standard early-curriculum exercise on Beer-Lambert and protein quantitation. Calculator handles arithmetic; students focus on the conceptual link between absorption, concentration, and molecular structure.

Technical Reference

Origin of Beer-Lambert. Pierre Bouguer (1729) first formulated the relation between transmitted light and pathlength for absorbing media. Johann Heinrich Lambert (1760) systematized it in his Photometria. August Beer (1852) extended it to relate absorption to concentration. The combined Beer-Lambert (or Beer-Lambert-Bouguer) law is now the foundation of all UV-Vis spectrophotometry. Modern double-beam spectrophotometers (Beckman DU-7, Cary, Shimadzu UV-2700, NanoDrop, etc.) implement it with ±1% accuracy across A = 0.05-2.0.

Why 280 nm for Proteins? Aromatic amino acids absorb in the UV: Trp (λmax 280 nm, ε 5,500); Tyr (λmax 274 nm, ε 1,490); Phe (λmax 257 nm, ε 200, mostly negligible at 280); cystine (disulfide, ε 125 at 280, broad). Their summed absorption at 280 nm is the protein A280 peak. The narrow specificity of this absorption to aromatic residues makes 280 nm convenient (no buffer interference at typical protein concentrations) but limits applicability to proteins WITH aromatic residues — a few proteins (e.g. amelogenin, certain elastin domains) lack Trp and Tyr and require Bradford / BCA / Lowry quantitation instead.

Edelhoch / Pace Extinction Coefficient Calculation. Edelhoch (1967, Biochemistry 6:1948) developed the empirical formula for predicting ε(280) from amino-acid composition. Pace, Vajdos, Fee, Grimsley & Gray (1995, Protein Sci. 4:2411) refined the parameters. Modern formula:

ε(280 nm, M⁻¹·cm⁻¹) = 5500 × n_Trp + 1490 × n_Tyr + 125 × n_cystine

Where n_cystine = number of disulfide bridges (NOT total cysteines; only oxidized cystines contribute). Reduced cysteine ε(280) ≈ 0. Accuracy: ±5% for properly folded proteins; deviations occur for proteins with unusual chromophores (heme, flavin, retinal) or non-standard amino acids.

Common Protein Reference Values.

  • BSA (bovine serum albumin): ε 43,824, MW 66,430. The 1 mg/mL = 0.66 A280 ratio is the famous BSA reference standard.
  • IgG (typical mouse/rabbit/human): ε 210,000, MW 150,000. 1 mg/mL ≈ A280 1.4.
  • Lysozyme (hen egg white): ε 38,000, MW 14,300. 1 mg/mL ≈ A280 2.66 (hyperabsorbent for its size due to high Trp content — 6 Trp residues).
  • BSA fraction V (lyophilized supplier-grade): typically 95-98% pure BSA + small amount of bovine globulins. Recover by ammonium sulfate fractionation.
  • Phycobiliprotein labels (PE, APC): high ε due to multiple covalent bilin chromophores per molecule. Standard for flow cytometry. PE: ε 1.96 × 10⁶ at 565 nm; APC: 700,000 at 650 nm.
  • Fluorescent proteins (GFP, mCherry, etc.): ε at fluorophore peak (488 for GFP, 587 for mCherry) is 30,000-100,000; lower than at 280 nm.

Common Pitfalls and How to Avoid Them.

  • Absorbance < 0.1: noise dominates; signal-to-noise < 10. Concentrate (Amicon ultrafiltration), use longer pathlength (1 cm vs 1 mm), or switch to Bradford/BCA.
  • Absorbance > 1.5: spectrophotometer non-linearity (stray light, detector saturation). Dilute and re-measure.
  • Aggregates / turbidity: light scatter inflates apparent A. Action: centrifuge sample (10,000g × 5 min) or filter (0.2 µm); subtract A320 from A280 to correct for scatter (small correction factor; works only for mild turbidity).
  • Nucleic-acid contamination: DNA/RNA absorb at 260 nm with ε ~50× protein. Diagnostic: A260/A280 ratio. Pure protein typically 0.5-0.7; pure DNA 1.8-2.0; pure RNA 2.0-2.2. Ratio > 1.0 indicates significant nucleic-acid contamination — reprocess (anion exchange, RNase/DNase digestion).
  • Detergents and reducing agents: SDS doesn't affect A280 (transparent at this wavelength); DTT and β-mercaptoethanol don't either. But protease inhibitors (PMSF, AEBSF) and some buffer components (EDTA at high conc.) can interfere.
  • Aromatic-residue-poor proteins: amelogenin, certain disordered proteins, structural elastins — A280 is weak. Use BCA or Lowry instead (these measure total protein via reactions with peptide bonds and side chains, not aromatic residues).

Alternative Protein Quantitation Methods.

  • Bradford assay (Coomassie blue): dye-binding to basic and aromatic residues, A595 measured. Sensitive (0.1-50 µg/mL). Sensitive to detergents; somewhat protein-dependent.
  • BCA (bicinchoninic acid): Cu²⁺ reduced to Cu⁺ by peptide bonds and certain residues; complexes with BCA → purple, A562. Sensitive (0.5-1500 µg/mL). Compatible with most detergents.
  • Lowry assay (Folin-Ciocalteu): oldest method (1951). Cu²⁺ reduces Folin reagent; complex absorbs at 660 nm. Sensitive (1-1000 µg/mL). Many interferences; mostly replaced by BCA.
  • NanoDrop UV-Vis: microvolume (1-2 µL), 0.1-1 cm pathlength options. Same Beer-Lambert math; convenient for small samples. Limit of quantitation ~0.05 mg/mL.

Modern Variations. NanoDrop and similar microvolume spectrophotometers automatically compute concentration when given protein type — same Beer-Lambert math, presets for common proteins. Multi-wavelength deconvolution can simultaneously quantify protein + label (degree of labeling for fluorescent conjugates). Mass spectrometry-based quantitation (intact-mass MS, or protein-specific peptide quantitation via SRM/MRM) is the gold standard for absolute quantitation but requires standards and instrumentation. References: Beer (1852); Lambert (1760); Bouguer (1729); Edelhoch (1967); Pace et al. (1995); Cytiva NanoDrop user guides; ExPASy ProtParam (Gasteiger et al., 2005).

Conclusion

Beer-Lambert UV-Vis is the workhorse of protein quantitation — fast, non-destructive, requires only ~1 µL of sample (NanoDrop) or ~50 µL (standard cuvette), and works across 4-5 orders of magnitude of concentration. The math is one formula: c = A / (ε × l), where ε is the molar extinction coefficient (auto-filled for 14 common proteins or computable from sequence via ExPASy ProtParam), A is measured absorbance at the protein's λmax (typically 280 nm), and l is cuvette pathlength (1 cm standard).

Three operational reminders: (1) Keep absorbance in the linear range (A = 0.1-1.5). Below 0.1 noise dominates; above 1.5 the spectrophotometer departs from linearity. Dilute or concentrate as needed. (2) Sample preparation matters more than the calculation: centrifuge or filter to remove aggregates/turbidity (scatter inflates A); blank against the actual buffer (matrix matching); check A260/A280 ratio < 1.0 for pure protein (higher indicates DNA/RNA contamination). (3) For unknown or engineered proteins, compute ε from sequence using ExPASy ProtParam (Edelhoch / Pace formula): ε(280) = 5500 × n_Trp + 1490 × n_Tyr + 125 × n_cystine. For crude samples or proteins with no aromatic residues, switch to Bradford / BCA / Lowry colorimetric assays.

Frequently Asked Questions

What is the Protein Concentration Calculator?
It implements the standard Beer-Lambert UV-Vis quantitation: c = A / (ε × l), where A is absorbance, ε is molar extinction coefficient (M⁻¹·cm⁻¹), and l is pathlength (cm). 14 protein presets with auto-filled ε and MW (BSA, IgG, lysozyme, insulin, RNase A, streptavidin, PE, APC, individual amino acids) plus custom mode. Output: protein concentration in 5 unit systems (M, mM, µM, mg/mL, µg/mL) with dilution-factor correction.

Pro Tip: Pair this with our Molarity Calculator.

What is the Beer-Lambert law?
A = ε × c × l, derived by Pierre Bouguer (1729), Johann Lambert (1760), and August Beer (1852). It states that absorbance (A) is proportional to molar extinction coefficient (ε), concentration (c), and pathlength (l). For protein quantitation: rearrange to c = A / (ε × l). Valid in the LINEAR RANGE (typically A = 0.1-1.5); below 0.1 noise dominates; above 1.5 the spectrophotometer enters non-linearity.
What is an extinction coefficient?
The molar absorptivity at a specific wavelength. Symbol ε; units M⁻¹·cm⁻¹ (alternatively L·mol⁻¹·cm⁻¹). For a 1 M solution in a 1 cm cuvette, ε equals the absorbance. For proteins, ε is dominated by aromatic residues (Trp, Tyr, cystine) and is computed from sequence via the Edelhoch / Pace formula: ε(280) = 5500 × n_Trp + 1490 × n_Tyr + 125 × n_cystine. Use ExPASy ProtParam (web.expasy.org/protparam) to compute for any protein from FASTA sequence.
Why is 280 nm used for proteins?
Because aromatic amino acids absorb maximally near 280 nm. Tryptophan (λmax 280 nm, ε 5,500), tyrosine (274 nm, 1,490), and disulfide-bridged cystine (broad, 125 at 280) dominate; phenylalanine (257 nm, 200) is mostly negligible. The 280 nm peak is selective for proteins (no buffer interference at typical concentrations) and is the universal reference wavelength for UV-Vis protein quantitation. Special chromophores (PE 565 nm, APC 650 nm, GFP 488 nm) absorb at their characteristic wavelengths instead.
What's the extinction coefficient of BSA?
43,824 M⁻¹·cm⁻¹ at 280 nm (some sources use 43,000 or 44,000; the exact value depends on reference and lot). MW = 66,430 g/mol. Practical reference: 1 mg/mL BSA gives A280 = 0.66 — the famous "BSA = 0.66 per mg/mL" rule of thumb in biochemistry labs. BSA is the most-used protein standard in laboratories worldwide for calibration curves and as a blocking agent.
What's the extinction coefficient of IgG?
~210,000 M⁻¹·cm⁻¹ at 280 nm (varies 200,000-220,000 by source). MW typical ~150,000 g/mol (160,000 for IgG3, 146,000 for IgG1). Practical reference: 1 mg/mL IgG gives A280 ≈ 1.4 (= 210,000 × 1 / 150,000 = 1.4 in Beer-Lambert). For ELISA / Western standards: prepare 1.0 mg/mL → working concentrations of 1, 10, 100 ng/mL via serial 10× dilutions.
How do I calculate ε for an unknown protein?
Use the Edelhoch / Pace formula: ε(280) = 5500 × n_Trp + 1490 × n_Tyr + 125 × n_cystine. Source: count Trp, Tyr, and cystine residues in the FASTA amino-acid sequence. Easiest: paste the sequence into ExPASy ProtParam (web.expasy.org/protparam/) — automatically computes ε, MW, pI, and other parameters. Accuracy: ±5% for properly folded proteins; deviations for proteins with unusual chromophores (heme, flavin, retinal) or aromatic-residue-poor proteins.
What absorbance range is OK for measurement?
A = 0.1 to 1.5 for accurate quantitation. Below 0.1: signal-to-noise becomes poor (instrument noise dominates). Above 1.5: spectrophotometer departs from linearity (stray light, detector saturation; Beer-Lambert breaks down). Optimal: aim for A = 0.5-1.0 for best precision (~1% RSD). If A is too low: concentrate sample, use longer pathlength, or switch to Bradford/BCA. If A is too high: dilute 5-10× and re-measure with appropriate dilution factor.
How do I deal with nucleic acid contamination?
Check the A260/A280 ratio. Pure protein: 0.5-0.7 (DNA absorbs only weakly at 280, mostly at 260). Pure DNA: 1.8-2.0. Pure RNA: 2.0-2.2. Ratio > 1.0: significant nucleic-acid contamination; reprocess via anion-exchange chromatography or DNase/RNase digestion. Quick correction (Warburg-Christian, approximate): protein concentration (mg/mL) ≈ 1.55 × A280 − 0.76 × A260 — works for crude lysates but not as accurate as proper purification + Beer-Lambert.
When should I use Bradford or BCA instead of UV-Vis?
Use Bradford / BCA when: (1) you don't know the protein (or it's a mixture); (2) the protein lacks aromatic residues (rare but happens — amelogenin, certain elastins); (3) sample is too dilute for A280 (< 50 µg/mL); (4) sample is contaminated with nucleic acids or other UV-absorbing species; (5) you need a small-volume colorimetric measurement and don't have a NanoDrop. Use UV-Vis when: you have pure protein with known ε; need a fast, non-destructive measurement; have NanoDrop access (only 1-2 µL needed); want absolute concentration without standard curves.
How does dilution factor work?
If you diluted the sample BEFORE measurement, multiply the in-cuvette concentration by the dilution factor. Example: stock too concentrated for direct measurement (A > 2). Take 10 µL stock + 90 µL buffer = 1:10 dilution = DF = 10. Measure A on the diluted sample. Calculator computes c_cuvette = A / (ε × l), then c_original = c_cuvette × 10. Common dilutions: 1:2 (DF 2), 1:5 (DF 5), 1:10 (DF 10), 1:100 (DF 100). Always pipette accurately for small volumes (use calibrated micropipette, ±1% tolerance).

Author Spotlight

The ToolsACE Team - ToolsACE.io Team

The ToolsACE Team

Our ToolsACE biology team built this calculator to handle the standard <strong>UV-Vis spectrophotometric protein quantitation</strong> via the Beer-Lambert law: <strong>c = A / (ε × l)</strong>, where A is measured absorbance, ε is the molar extinction coefficient (M⁻¹·cm⁻¹), and l is the cuvette pathlength (cm). The calculator includes <strong>14 protein presets</strong> with auto-filled extinction coefficients and molecular weights — IgG (210,000 / 150,000), BSA (43,824 / 66,430), lysozyme (38,000 / 14,300), insulin (5,840 / 5,808), RNase A (9,800 / 13,700), streptavidin (167,760 / 53,000), phycoerythrin (1,960,000 / 240,000), allophycocyanin (700,000 / 105,000), individual amino acids (Trp, Tyr, Phe, Cys), ATP, and aldose. <strong>Custom protein</strong> mode for any user-supplied ε and MW. Multi-unit pathlength input (cm / mm / m); dilution-factor field automatically corrects for sample dilution before measurement. <strong>Output:</strong> protein concentration in 5 unit systems (M, mM, µM, mg/mL, µg/mL) with full transparent calculation breakdown. <strong>Smart warnings</strong> flag absorbance outside the linear quantitation range (0.1-1.5), exceptional ε values, and signal-to-noise concerns.

ExPASy ProtParam (Edelhoch / Pace) extinction coefficient calculationsStandard biochemistry references; CRC Handbook of Chemistry and PhysicsThermoFisher / Cytiva NanoDrop protein quantitation protocols

Disclaimer

Beer-Lambert quantitation requires linear-range absorbance (typically 0.1-1.5 AU). Below 0.1, signal-to-noise becomes poor; above 1.5, the spectrophotometer enters non-linearity. Sample preparation matters: aggregates and turbidity scatter light → falsely high A; nucleic-acid contamination absorbs at 260 nm and bleeds into 280 nm (use A260/A280 ratio < 1.0 indicates pure protein). Detergents and reducing agents can interfere; for crude lysates use Bradford, BCA, or Lowry assays. Extinction coefficients in the preset list are typical published values; for unknown proteins, calculate from sequence using ExPASy ProtParam (Edelhoch/Pace formula: ε(280) = 5500 × n_Trp + 1490 × n_Tyr + 125 × n_cystine). Bradford / BCA / Lowry colorimetric assays sidestep ε requirements but require a standard curve and are prone to interferences. References: ExPASy ProtParam; Edelhoch (1967); Pace et al. (1995); Cytiva / GE NanoDrop protein protocols; CRC Handbook of Chemistry and Physics.