Convert your A280 reading into protein concentration in mg/mL or uM with blank correction, dilution, and the coefficient style you already have.
Advanced options
How to use our Protein Concentration Calculator
- Choose "What do you know about your protein?" based on the number you already have: molar extinction coefficient, 1% absorptivity at 280 nm (E1%), or the quick estimate.
- Enter "Absorbance at 280 nm (A280)", "Path length (cm)", "Extinction coefficient at 280 nm" when needed, and "Molecular weight (kDa)" if you want both mg/mL and uM.
- Open "Advanced options" if needed and enter "Blank absorbance at 280 nm (A280)" and "Dilution factor" so the result reflects the original stock sample.
- Click Calculate, then check "Method used" and "Corrected A280 used" first to confirm the calculator used the method you expected and that the corrected absorbance is positive.
- Use "Protein concentration" in mg/mL for stock prep and assay setup, and use "Protein concentration" in uM when you need molecule-based concentration for binding, enzyme, or labeling work.
Definitions
A280: Absorbance measured at 280 nm, a UV wavelength often used to estimate protein concentration.
Path length (cm): The distance light travels through the sample. A standard cuvette is often 1 cm.
Extinction coefficient at 280 nm: A protein-specific number that tells how strongly the protein absorbs UV light at 280 nm. In molar mode it is epsilon in M^-1 cm^-1. In E1% mode it is absorptivity for a 1% solution in cm^-1.
Molecular weight (kDa): The size of one mole of your protein. It is used to convert between molar concentration and mass concentration.
Blank absorbance at 280 nm (A280): The background absorbance from buffer or solvent that should be subtracted from the sample reading.
Dilution factor: How much the sample was diluted before reading. A value of 10 means the original sample was 10 times more concentrated than the measured diluted sample.
mg/mL: Milligrams of protein per milliliter of solution, a mass concentration.
uM: Micromolar, or millionths of a mole per liter, a molecule-based concentration unit.
E1%: The absorbance behavior of a 1% protein solution, where 1% means 1 g per 100 mL.
Common mistakes and quick fixes
Mistake: Using "Absorbance at 280 nm (A280)" that was already blank-corrected, then also entering a nonzero "Blank absorbance at 280 nm (A280)".
Fix: If your instrument already blanked the sample, leave "Blank absorbance at 280 nm (A280)" at 0.000 so "Corrected A280 used" does not get subtracted twice.
Mistake: Leaving "Path length (cm)" at 1 when your microvolume or plate reader used a shorter path.
Fix: Enter the actual "Path length (cm)" reported by your instrument, or your "Protein concentration" can be too high or too low.
Mistake: Entering an E1% value while "What do you know about your protein?" is set to molar extinction coefficient, or the reverse.
Fix: Match the number in "Extinction coefficient at 280 nm" to the selected method in "What do you know about your protein?" before you trust "Method used" and the concentration outputs.
Mistake: Forgetting to enter the sample dilution in "Dilution factor".
Fix: Enter 1 for no dilution, or enter the full fold dilution so "Protein concentration" reports the original sample instead of the diluted reading.
Mistake: Using the wrong "Molecular weight (kDa)" and then relying on "Protein concentration" in uM.
Fix: Check the protein's actual "Molecular weight (kDa)" because the uM result depends directly on it.
Mistake: Ignoring a zero or negative "Corrected A280 used" value.
Fix: Recheck "Absorbance at 280 nm (A280)" and "Blank absorbance at 280 nm (A280)" because a nonpositive corrected reading cannot support a positive protein concentration here.
Limitations & Key Assumptions / Boundary Conditions
- This calculator uses the A280 Beer-Lambert approach, which works best for reasonably pure proteins with absorbance driven by amino acids such as tryptophan and tyrosine.
- If "Corrected A280 used" is zero or negative, the result is treated as invalid instead of forcing a positive concentration.
- The quick estimate mode assumes 1 A280 equals about 1 mg/mL at a 1 cm path length, so it is only a rough shortcut and not protein-specific.
- In molar extinction coefficient mode, uM does not require molecular weight; mg/mL does. In E1% and quick estimate modes, mg/mL does not require molecular weight; uM does. Leave "Molecular weight (kDa)" blank if it is unknown.
- Very high absorbance can fall outside an instrument's linear range, so the math may still run while the real reading is less reliable.
- The calculator uses the entered path length exactly. If your instrument auto-normalized to 1 cm and you also enter a shorter path, you can overcorrect the result.
- Hidden mode-specific assumptions are not mixed across modes: only the selected coefficient style is used in the calculation.
Methodology
Core calculation flow
The calculator first finds the absorbance that actually came from the protein sample by subtracting the blank from the sample reading. It then applies the selected coefficient method and finally adjusts for any dilution so the answer reflects the original sample.
A = ε (molar extinction coefficient) x l (path length in cm) x c (molar concentration)
A_corr = A280_sample - A280_blank
Beer-Lambert law says absorbance is proportional to concentration, path length, and extinction coefficient [1][2].
Molar extinction coefficient mode
Use this mode when your protein sheet gives epsilon in M^-1 cm^-1. The calculator finds molar concentration first, then converts to mg/mL if molecular weight is available.
c_stock_M = (A_corr / (epsilon x l)) x DF
conc_mg_per_mL = c_stock_M x MW_g_per_mol
conc_uM = c_stock_M x 1000000
Because 1 g/L equals 1 mg/mL numerically, multiplying molar concentration in mol/L by molecular weight in g/mol gives a convenient mg/mL value.
E1% mode
Use this mode when you have a 1% absorptivity value instead of molar epsilon. This method gives mg/mL directly, then converts to uM if molecular weight is available.
conc_mg_per_mL = (A_corr / (E1pct x l)) x 10 x DF
conc_uM = (conc_mg_per_mL / MW_g_per_mol) x 1000000
The factor of 10 comes from the definition of a 1% solution:
1 g/100 mL = 10 mg/mL.
Quick estimate mode
This shortcut assumes a typical protein gives about 1 mg/mL for an A280 of 1.0 at a 1 cm path length. It is fast, but it is not protein-specific.
conc_mg_per_mL = (A_corr / l) x DF
conc_uM = (conc_mg_per_mL / MW_g_per_mol) x 1000000
Worked mini-example
Suppose you choose molar mode, enter A280 = 0.9, blank = 0.1, path length = 1 cm, extinction coefficient = 45000, molecular weight = 60 kDa, and dilution factor = 5.
A_corr = 0.9 - 0.1 = 0.8
c_stock_M = (0.8 / (45000 x 1)) x 5 = 0.0000888889 M
conc_uM = 0.0000888889 x 1000000 = 88.8889 uM
MW_g_per_mol = 60 x 1000 = 60000 g/mol
conc_mg_per_mL = 0.0000888889 x 60000 = 5.3333 mg/mL
That means the original stock sample is about 5.33 mg/mL, or about 88.89 uM.
Assumptions that affect real results
The method assumes your A280 reading is in a usable linear range, your sample is blanked correctly, and the coefficient matches your actual protein. Real samples can differ if they contain contaminants, if the protein is not pure, or if the entered molecular weight or path length is wrong.