Protein Concentration Calculator
Calculate protein concentration with our free science calculator. Uses standard scientific formulas with unit conversions and explanations.
Formula
c = A / (epsilon x l)
Where c = molar concentration (M), A = absorbance at 280 nm, epsilon = molar extinction coefficient (M-1 cm-1), and l = cuvette path length (cm). Multiply by molecular weight and dilution factor to get mg/mL concentration.
Worked Examples
Example 1: BSA Concentration from A280 Reading
Problem: A BSA sample (MW = 66,500 Da, epsilon = 43,824 M-1 cm-1) gives an A280 of 0.45 in a 1 cm cuvette after 10x dilution. Find the stock concentration.
Solution: c = A / (epsilon x l) = 0.45 / (43824 x 1) = 1.027 x 10^-5 M\nConcentration = 1.027 x 10^-5 x 66500 / 1000 = 0.683 mg/mL\nAdjusted for 10x dilution: 0.683 x 10 = 6.83 mg/mL
Result: Stock concentration: 6.83 mg/mL (1.027 x 10^-4 M)
Example 2: Total Protein in a Sample Volume
Problem: An antibody solution (MW = 150,000 Da, epsilon = 210,000 M-1 cm-1) reads A280 = 0.80 in 1 cm path. No dilution. Volume = 200 uL.
Solution: c = 0.80 / (210000 x 1) = 3.81 x 10^-6 M\nConcentration = 3.81 x 10^-6 x 150000 / 1000 = 0.571 mg/mL\nTotal protein = 0.571 x (200/1000) = 0.114 mg = 114 ug
Result: Concentration: 0.571 mg/mL | Total protein: 114 ug in 200 uL
Frequently Asked Questions
How does Beer-Lambert Law apply to protein concentration measurement?
The Beer-Lambert Law (A = epsilon x l x c) is the foundational equation for spectrophotometric protein concentration measurement. It states that absorbance (A) is directly proportional to the molar extinction coefficient (epsilon), the path length of the cuvette (l, usually 1 cm), and the concentration of the analyte (c). For proteins, absorbance is typically measured at 280 nm (A280), where tryptophan and tyrosine residues absorb UV light. By rearranging the equation to c = A / (epsilon x l), you can calculate the molar concentration directly from the measured absorbance. This method is non-destructive, fast, and requires no reagents.
Why is the dilution factor important in protein concentration calculations?
The dilution factor corrects for any dilution performed on the original sample before spectrophotometric measurement. Proteins often need to be diluted so that their absorbance falls within the linear range of Beer-Lambert Law (typically A280 between 0.1 and 1.0). If you dilute a sample 10-fold before measuring, you must multiply the calculated concentration by 10 to obtain the true concentration of the original stock solution. Failing to account for dilution is one of the most common errors in protein quantification. Always record the exact dilution ratio, as even small errors propagate through all downstream calculations and experimental protocols.
What are common methods for measuring protein concentration besides A280?
Several alternative methods exist for protein quantification. The Bradford assay uses Coomassie Brilliant Blue dye binding and measures absorbance at 595 nm, with a linear range of 1-25 micrograms per milliliter. The BCA (bicinchoninic acid) assay detects reduced copper ions and is less sensitive to detergents. The Lowry assay combines Biuret reaction with Folin-Ciocalteu reagent reduction. Fluorometric assays using reagents like NanoOrange or CBQCA offer high sensitivity down to nanogram levels. Each method has advantages: A280 is fastest and non-destructive, Bradford is robust and simple, BCA is compatible with detergents, and fluorometric methods provide the greatest sensitivity.
What factors can interfere with accurate protein concentration measurements?
Multiple factors can introduce errors in spectrophotometric protein measurements. Nucleic acid contamination absorbs strongly at 260 nm and can inflate A280 readings; an A260/A280 ratio above 0.6 suggests contamination. Buffer components such as imidazole, DTT, and certain detergents absorb at 280 nm and create false high readings. Light scattering from aggregated proteins or particulates increases apparent absorbance. Temperature fluctuations affect both protein solubility and spectrophotometer readings. Using the wrong extinction coefficient, forgetting dilution factors, or working outside the linear range of Beer-Lambert Law (absorbance above 1.0) are common procedural errors that significantly impact accuracy.
Can I use the results for professional or academic purposes?
You may use the results for reference and educational purposes. For professional reports, academic papers, or critical decisions, we recommend verifying outputs against peer-reviewed sources or consulting a qualified expert in the relevant field.
How do I get the most accurate result?
Enter values as precisely as possible using the correct units for each field. Check that you have selected the right unit (e.g. kilograms vs pounds, meters vs feet) before calculating. Rounding inputs early can reduce output precision.