Fit a linear protein assay curve from your own standards, then interpolate unknown samples only within the measured response range.
Leave this off when the values you enter are already blank-corrected or reported as net absorbance by your instrument or analysis software. Turn it on only for raw readings. Do not subtract the same blank twice; instrument zeroing against water or a reference is not necessarily the same as subtracting the matrix-matched zero-protein blank required by your protocol.
Linear fit only. Bradford response can curve across a broad range and can differ between proteins. Use this calculator only for the range your kit instructions or validated assay procedure identifies as linear; use a manufacturer-specified nonlinear model when needed.
Bradford — A595 changes the reading label and guidance only. This calculator does not supply assay-specific working ranges, standards, or conversion factors.
Enter one known protein concentration per row, with up to 3 A595 readings. The calculator fills in the mean and SD.
Incomplete rows are ignored. At least three included points with three distinct concentrations are required.
| Use in fit | Known protein concentration (mg/mL) | Replicate A595 readings | Calculated from readings | Remove | |||
|---|---|---|---|---|---|---|---|
| Reading 1 | Reading 2 | Reading 3 | Mean A595 | SD | |||
| — | — | ||||||
| — | — | ||||||
| — | — | ||||||
The illustrative values are non-experimental and are not a recommended curve for any assay or protocol.
Include at least three complete standards to fit the curve.
A dilution factor of 10 means a 1:10 dilution. Original concentration = interpolated concentration × dilution factor.
| Sample | Reading 1 | Reading 2 | Reading 3 | Dilution factor | Mean A595 | Diluted sample (mg/mL) | Original sample (mg/mL) | Remove |
|---|---|---|---|---|---|---|---|---|
| — | — | — |
A colorimetric protein result is an interpolation against standards measured in the same run. This calculator fits a straight line through the linear portion of those standards; it does not replace the assay kit protocol or decide whether a particular fit is suitable.
For routine relative quantitation, BSA or bovine gamma globulin are commonly used reference proteins. The result is relative to that chosen standard: different proteins can produce different color responses in Bradford, BCA, and Lowry methods. When accuracy for one purified target protein matters, a standard prepared from that target is the stronger choice.
Prepare standards in the same diluent or closely matched matrix as the unknowns whenever practical. Matching the buffer, detergent content, and other sample components reduces the risk that an apparent concentration difference is actually a matrix effect.
Use the mean reading from the appropriate zero-protein blank when your protocol calls for blank correction, then apply that same correction to every standard and unknown. If the instrument or analysis software has already exported blank-corrected, net absorbance values, enter those values directly and leave the calculator's blank-correction option off. Enter replicate readings individually so the calculator can show their mean and sample standard deviation. Replicates reveal pipetting or plate-position variation that a single measurement conceals.
The calculator accepts one to three readings per row and requires at least three included concentration levels to calculate a line. Your own kit instructions or SOP should determine the number, spacing, and placement of standards.
Do not assume that every stated assay range is linear. Bradford response can vary with protein composition, and some BCA methods specify a nonlinear curve-fitting approach over their full range. This page deliberately uses only least-squares linear regression. Restrict the entered standards to a range validated as linear for the exact assay format, reagent volumes, incubation conditions, reader, and standard protein you used.
R² describes how closely the entered standard means follow the fitted line; it does not prove that the chemistry, sample matrix, or selected range is valid. Review the plot, replicate variation, blanks, and protocol controls rather than using R² alone as a pass/fail criterion.
The calculator converts each unknown's mean corrected absorbance to the concentration in the measured, diluted aliquot. If that aliquot was diluted before the assay, multiply by the dilution factor to report the original sample concentration. For example, an interpolated 0.40 mg/mL result from a 1:5 dilution corresponds to 2.0 mg/mL in the original sample.
Results are reported in mg/mL because the standard table uses mg/mL. Convert any protocol concentrations before entry: 1,000 µg/mL equals 1 mg/mL. Do not report a result outside the entered calibration range. Dilute samples above range and rerun them; treat samples below range as not quantified by that curve.
The following values are the same non-experimental values available through Load illustrative example. They demonstrate why raw and blank-corrected values must not be mixed. A mean blank of 0.05 is subtracted from every reading.
Raw A595 = 0.504 × concentration + 0.055333
Corrected A595 = raw A595 − 0.05 = 0.504 × concentration + 0.005333
Unknown raw A595 = 0.56; corrected A595 = 0.56 − 0.05 = 0.51
Concentration = (corrected A595 − intercept) / slope = (0.51 − 0.005333) / 0.504 = 1.001 mg/mL
For a 1:5 dilution: original concentration = 1.001 × 5 = 5.01 mg/mL
The 1.001 mg/mL result is the concentration in the diluted aliquot. The calculator reports the same value before applying the entered dilution factor.
Assay chemistry matters. Bradford assays can be affected by detergents and some basic buffers, while standard BCA methods can be affected by reducing agents and metal-chelating components. The relevant kit documentation, including its compatibility table and instructions for a matched blank, takes priority over generic advice. If the matrix is incompatible, use a validated cleanup, dilution, or alternative assay strategy rather than relying on a mathematical correction.
BCA, Bradford, and Lowry assays estimate concentration from a color response relative to a reference protein. A280 estimates use direct UV absorbance and an extinction coefficient. They answer different experimental questions and have different interferences. For direct UV measurements, use the A280 Protein Concentration Calculator. To convert a measured mass concentration to molarity, use the Protein Molarity Calculator.
Three distinct, complete concentration levels are the mathematical minimum for this calculator. Use the number and spacing required by the specific kit or SOP; additional standards are often useful for defining the chosen working range.
No. An absorbance above range should be measured again after a suitable dilution. A reading below range is not a quantified concentration from that curve.
A factor of 10 represents a tenfold, or 1:10, pre-assay dilution. The calculator multiplies the concentration measured in that diluted aliquot by 10 to estimate the original sample concentration.
No. A high R² can coexist with an unsuitable standard protein, an interfering matrix, poorly controlled incubation, or a curve that should not be modeled linearly. Use it as one diagnostic alongside protocol controls and replicate quality.