Protein Concentration Calculator
Determines protein concentration from a Bradford assay absorbance reading using your standard curve's slope and intercept. Essential for normalizing samples before Western blots, ELISAs, or enzymatic assays.
About this calculator
The Bradford assay estimates protein concentration by measuring the absorbance of a sample at 595 nm after binding Coomassie Brilliant Blue G-250 dye. The dye shifts from red to blue upon binding protein, and the absorbance at 595 nm is proportional to protein content. To convert absorbance to concentration, you first generate a standard curve using known concentrations of a reference protein (typically BSA), then fit a linear regression to obtain a slope (m) and y-intercept (b). The formula is: Concentration = (A₅₉₅ − b) / m, which is simply rearranging the linear equation A = m·C + b for concentration. This approach corrects for background absorbance captured in the intercept and scales the reading according to your specific reagent batch and instrument, making results reproducible across labs.
How to use
Suppose your BSA standard curve gives a slope of 0.85 A595 per mg/mL and a y-intercept of 0.02. You measure an unknown sample and read an absorbance of 0.614 A595. Apply the formula: Concentration = (0.614 − 0.02) / 0.85 = 0.594 / 0.85 ≈ 0.699 mg/mL. If you diluted your sample 5-fold before the assay, multiply by the dilution factor: 0.699 × 5 = 3.495 mg/mL in the original sample. You would then use this value to load equal protein masses across all lanes of your gel.
Frequently asked questions
Why do I need a standard curve for the Bradford assay?
Coomassie dye response is not perfectly linear across all protein concentrations, and the slope of the absorbance-versus-concentration relationship varies with dye lot, reagent age, and spectrophotometer model. A freshly prepared standard curve using your exact reagents on the same day as your unknowns accounts for all these variables. Without it, applying a generic conversion factor can introduce errors of 20% or more. Always include standards bracketing your expected sample concentrations to keep measurements within the validated linear range of the assay.
What is the linear range of the Bradford assay and why does it matter?
The standard Bradford assay is typically linear between about 0.2 and 1.4 mg/mL BSA-equivalent protein, while the microassay version extends sensitivity down to 1–25 µg/mL. If your sample falls outside this range, the relationship between absorbance and concentration becomes nonlinear, and the simple slope-intercept formula no longer applies. Samples too concentrated should be diluted and reassayed; samples too dilute may require the more sensitive microassay format. Reporting a concentration derived from an out-of-range reading is a common source of experimental error.
How does the Bradford assay compare to the BCA assay for measuring protein concentration?
Both assays are colorimetric and require a standard curve, but they differ in chemistry and interference profiles. The Bradford assay is faster (about 5 minutes of incubation) and is less susceptible to reducing agents like DTT, but it is sensitive to detergents such as SDS and Triton X-100. The BCA (bicinchoninic acid) assay tolerates detergents better and has a wider linear range, but it is inhibited by reducing agents and requires a longer incubation at elevated temperature. Choosing between them depends on what is in your sample buffer — check reagent compatibility before selecting your assay method.