Protein Concentration Calculator
Enter your absorbance reading, extinction coefficient, and path length to get protein concentration in mg/mL, µg/µL, µM, and nmol/mL in seconds. Switch between three methods: Beer-Lambert (A280), standard-curve assay (Bradford, BCA, Lowry), or dilution math (C1V1 = C2V2). Pick a preset protein such as BSA or IgG to auto-fill the extinction coefficient and molecular weight, or enter your own values for any custom protein.
Formula
Worked example
BSA in a 1 cm cuvette: A280 = 0.84, blank = 0.00, epsilon = 43,824 M-1cm-1, MW = 66,463 g/mol, dilution factor = 10. Corrected absorbance: 0.84. Molar concentration in cuvette: 0.84 / (43,824 x 1) = 1.917e-5 M. In mg/mL: 1.917e-5 x 66,463 = 1.274 mg/mL. With DF = 10, original = 12.74 mg/mL.
How protein concentration is measured
Three methods dominate modern protein quantification. The UV absorbance method (A280) exploits the fact that the aromatic amino acids tryptophan and tyrosine absorb light at 280 nm according to the Beer-Lambert law: A = epsilon x c x l, where epsilon is the molar extinction coefficient, c is concentration, and l is the path length. This method is quick, non-destructive, and needs no reagents, but it requires knowing the protein-specific extinction coefficient and is sensitive to nucleic acid contamination. Colorimetric assays such as Bradford (Coomassie G-250 dye), BCA (bicinchoninic acid), and Lowry measure a colour change proportional to protein concentration against a standard curve, typically built from BSA. Bradford is fast and compatible with most buffers; BCA tolerates more contaminants and detergents; Lowry is sensitive but slow. The third approach, dilution math using C1V1 = C2V2, is not a measurement method but a preparation tool: given a known stock concentration, it tells you how much stock to dilute into what total volume to reach a target concentration.
Beer-Lambert law and the extinction coefficient
The extinction coefficient (also called the molar attenuation coefficient, epsilon) is a fixed physical property of each protein at a given wavelength. It quantifies how strongly the protein absorbs light per unit concentration and per unit path length. At 280 nm, the dominant contributors are tryptophan (epsilon per residue approx. 5,500 M-1cm-1) and tyrosine (approx. 1,490 M-1cm-1), with a smaller contribution from disulfide bonds. Proteins without tryptophan, such as short peptides or some engineered proteins, may have very low A280 and are better quantified by colorimetric methods. If you do not know your protein's extinction coefficient, the ExPASy ProtParam tool can calculate it from the amino acid sequence using the Pace et al. method. Typical values range from around 5,000 M-1cm-1 for small tryptophan-free peptides to over 200,000 M-1cm-1 for large multi-tryptophan proteins like IgG. For reliable Beer-Lambert results, keep A280 between 0.05 and 1.5; readings outside this range tend to deviate from linearity.
Standard curve assays: Bradford, BCA, and Lowry
Colorimetric assays do not use absolute physical constants; instead they compare an unknown sample against a series of calibration standards of known concentration, usually BSA. You measure the absorbance (or fluorescence) of each standard, fit a straight line (y = m x C + b), and then invert the equation to find concentration from signal: C = (y - b) / m. This calculator accepts any slope and intercept, so it works for Bradford (typically measured at 595 nm), BCA (562 nm), Lowry (750 nm), or any other linear colorimetric assay. Important caveats: Bradford is incompatible with high detergent concentrations (SDS, Triton) and strong reducing agents. BCA is affected by reducing agents like DTT and beta-mercaptoethanol. Lowry is more sensitive than Bradford but slower and more sensitive to interfering compounds. Always match the buffer of your standards to your sample to minimise matrix effects, and ensure all sample readings fall within the linear portion of the standard curve.
Choosing the right output unit for your experiment
This calculator reports concentration in four interchangeable units. mg/mL (equal to g/L) is the most common unit in protein biochemistry and is suitable for gel loading, SDS-PAGE, Western blot, ELISA, and most biological assays. µg/µL is numerically identical to mg/mL and is favoured when thinking about pipette volumes (for example, 1 µg/µL means 1 µg per microlitre). Molar concentration in µM (micromolar) is essential for stoichiometric calculations, enzyme kinetics (Michaelis-Menten, Ki, kcat), binding affinities (Kd), and whenever you need to know the number of molecules. To convert between mass and molar concentration you need the molecular weight: c (mg/mL) = c (M) x MW (g/mol). nmol/mL is numerically identical to µM (1 µM = 1 nmol/mL) and appears in some enzyme activity reports. If your protein has multiple subunits, decide whether your molecular weight refers to the monomer or the assembled oligomer, as this changes the molar concentration by the oligomer number.
Common protein extinction coefficients and molecular weights
| Protein | Extinction coefficient (M⁻¹ cm⁻¹) | Molecular weight (g/mol) | Typical use |
|---|---|---|---|
| BSA (Bovine Serum Albumin) | 43,824 | 66,463 | Universal standard |
| IgG (Human/Rabbit) | 210,000 | 150,000 | Antibody quantification |
| Lysozyme | 37,970 | 14,313 | Activity assays, crystallography |
| Insulin | 6,200 | 5,808 | Hormone studies, folding assays |
| RNase A | 9,800 | 13,700 | RNA degradation assays |
| Streptavidin | 101,000 | 52,800 | Biotin-binding assays |
| GFP | 55,000 | 26,900 | Fluorescent tag quantification |
| HSA (Human Serum Albumin) | 35,700 | 66,500 | Clinical and binding assays |
Molar extinction coefficients at 280 nm and molecular weights for proteins commonly used as standards. Sources: ExPASy ProtParam, Sigma-Aldrich.
Frequently asked questions
What is a typical protein concentration for downstream experiments?
It varies widely by application. For SDS-PAGE and Western blot, loading 1-50 µg total protein per lane typically requires stock concentrations of 0.5-5 mg/mL. ELISA capture antibodies are often coated at 1-10 µg/mL. Crystallography trials commonly start at 5-20 mg/mL. ITC and SPR binding assays may need 0.1-1 µM (which could be very low mg/mL for a small protein). Always check the recommended range for your specific protocol.
Why does my A280 method give a different result than Bradford?
These methods measure different things. A280 uses the actual UV absorption of aromatic residues and depends on the protein-specific extinction coefficient. Bradford measures dye binding to basic and hydrophobic residues, and the response differs between proteins. BSA-based Bradford standards may over- or under-estimate your protein by 10-50% if your protein has a different amino acid composition. Using the same protein as a standard, or cross-checking with a known reference, reduces this discrepancy. Nucleic acid contamination (absorbs at 280 nm) can also inflate A280 results; Bradford is not affected by DNA.
How do I correct for nucleic acid contamination in the A280 reading?
The Warburg-Christian or Kalb-Bernlohr correction uses readings at both 280 nm and 260 nm to estimate protein concentration while accounting for nucleic acid absorption: protein (mg/mL) approximately equals 1.55 x A280 minus 0.76 x A260. This correction is approximate and assumes the nucleic acid has an average composition. For high nucleic acid contamination, DNase/RNase treatment or purification steps give more accurate results. The 260/280 ratio is also a quick purity indicator: a ratio below 0.6 suggests little nucleic acid contamination for a typical protein.
What is a dilution factor and how do I apply it?
A dilution factor is the ratio by which you diluted the sample before measurement. If you mixed 10 µL of your protein with 90 µL of buffer, your dilution factor is 10 (because the total volume is ten times the sample volume). The concentration you calculate from the measurement applies to the diluted solution; multiply by the dilution factor to recover the concentration in the original undiluted sample. For example, measuring 0.5 mg/mL after a 1:10 dilution means the stock was 5 mg/mL.
Why is path length important and when does it differ from 1 cm?
Path length (l) is the distance light travels through the sample. Standard cuvettes have a 1 cm path length, so Beer-Lambert calculations typically assume l = 1. Microvolume instruments like the NanoDrop use a very short path length (typically 0.05-1 mm, set automatically by the sample volume), which allows measurement of small volumes without dilution but requires the instrument to report A280 corrected to a 1 cm equivalent. If you are entering raw absorbance from a microvolume instrument, confirm whether it has already normalised to 1 cm (most do). If using a 0.5 cm cuvette, set the path length to 0.5 in this calculator.
Can I use this calculator for peptide concentrations?
Yes, provided you know the correct extinction coefficient and molecular weight for your peptide. Many short peptides lack tryptophan and have very low or zero absorption at 280 nm, making A280 unreliable. In that case, use a colorimetric assay (Bradford, BCA), measure at 205 nm (peptide bond absorption, but requires a UV-grade cuvette and is sensitive to many buffers), or use amino acid analysis. If your peptide contains tryptophan or tyrosine, ExPASy ProtParam can calculate the theoretical epsilon from its sequence.