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BCA Protein Assay Calculator (Standard Curve and Sample Concentration)

Enter the A562 readings for your BSA standards and samples (separate replicates with commas) and get the curve equation, R² and each sample's protein concentration with the dilution factor applied.

Inputs

Curve fit

BSA standards

Conc. (μg/mL)A562

Separate replicates with commas or spaces; they are averaged.

Results

A = 0.000869 + 0.000844 × C − 1.11×10⁻⁷ × C²

R²
0.99997
Blank
0.095
Standard range
25 to 2000 μg/mL

Samples

NameA562Dilution factorOn curveSample conc.
591 μg/mL2.96 mg/mL
395 μg/mL1.98 mg/mL

Formula

Blank: A = A562(well) − mean A562(0 μg/mL)

Quadratic: A = a + b × C + c × C²

Linear: A = a + b × C, C = (A − a) ÷ b

Sample concentration = curve concentration × dilution factor

Tip: Run standards and samples on the same plate, incubate them together and read 562 nm at the same time. Samples outside the standard range get no result; adjust the dilution and rerun.

You got this step right, but a whole experiment has dozens of linked parameters. Change one dilution factor and everything downstream has to be recalculated. In Vara the parameters of your whole experiment are connected, so changing one recalculates the rest automatically. Manage 3 experiments for free.

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How the formula works

  1. Prepare the working reagent: mix reagent A and reagent B at 50:1. It turns cloudy when B is added and clears to green after mixing.
  2. In a microplate, add 25 μL of standard or sample and 200 μL of working reagent per well, incubate at 37 °C for 30 minutes, cool to room temperature and read at 562 nm.
  3. Subtract the blank: average the replicates, then subtract the mean of the 0 μg/mL wells from every well.
  4. Fit absorbance A against concentration C using the blank-corrected standards: linear A = a + b × C, or quadratic A = a + b × C + c × C².
  5. Solve the curve for each sample's A. For a quadratic, take the root on the rising part of the curve.
  6. The result is the concentration in the well; multiply by the dilution factor used before the assay to get the original sample concentration.

Worked examples

Example 1: quadratic fit for a lysate

  1. Nine standards in duplicate; the 0 μg/mL wells read 0.094 and 0.096, so the blank is 0.095.
  2. Sample A was diluted 5-fold before the assay and reads 0.552 and 0.560: mean 0.556, 0.461 after blank subtraction.
  3. Quadratic fit: A = 0.000869 + 0.000844 × C − 1.11×10⁻⁷ × C², R² = 0.99997.
  4. Solving for A = 0.461 gives a curve concentration of 591 μg/mL.
  5. Original sample = 591 μg/mL × 5 = 2.96 mg/mL.

Example 2: the same data with a linear fit

  1. Linear fit: A = 0.0549 + 0.000627 × C, R² = 0.99009, which also looks high.
  2. Sample A now reads 648 μg/mL on the curve, 3.24 mg/mL in the original sample, about 10% higher than the quadratic.
  3. The difference comes from the bend at the high end: the 1500 and 2000 μg/mL points flatten the line and shift mid-range readings.

Example 3: how much to load for 20 μg of protein

  1. From example 1 the sample is 2.96 mg/mL, which is 2.96 μg of protein per μL.
  2. For 20 μg: 20 ÷ 2.96 ≈ 6.76 μL of lysate, then top every lane up to the same volume with loading buffer.

Common mistakes

Reading standards and samples at different times

BCA has no endpoint and the color keeps changing. If the standards are read first and the samples later, they correspond to different development times and the samples come out systematically high.

Extrapolating beyond the curve

Readings above 2000 μg/mL or below the lowest standard fall outside the curve and extrapolated values are unreliable. Change the dilution and rerun.

Choosing a straight line because R² looks good

In this page's default example, a full-range linear fit has R² 0.990, yet the sample comes out about 10% higher than with the quadratic. A high R² doesn't mean mid-range readings are right; use a quadratic for the full range, or only the standards in the linear part.

Standards and samples in different buffers

Reducing agents (DTT, β-mercaptoethanol) and chelators (EDTA) in lysis buffer interfere with color development. Standards made in water don't match samples in a lysis buffer that contains them. Prepare the standards in the same lysis buffer as the samples.

Forgetting the dilution factor

The curve gives the concentration in the well. If you diluted 5-fold before the assay, the original sample is 5 times the reading; skipping this means loading 5 times too much protein.

FAQ

Which BSA concentrations should a BCA standard curve use?+
The Thermo Pierce BCA (23225) microplate protocol uses nine BSA standards: 2000, 1500, 1000, 750, 500, 250, 125, 25 and 0 μg/mL, with 0 μg/mL as the blank. The standard protocol's working range is 20 to 2000 μg/mL. If you don't know roughly how concentrated your samples are, run two or three dilutions of each.
Should I subtract the blank, and how?+
Yes. Subtract the mean A562 of the 0 μg/mL wells from every standard and sample well, then fit the curve to the blank-corrected standards; the 0 point itself drops out of the fit. Tick "Subtract blank" and the calculator does this for you. Make the blank in the same buffer or lysis buffer as your samples.
Linear or quadratic fit?+
When the standards cover the full 25 to 2000 μg/mL range, the BCA curve bends at the high end, so a straight line misreads mid-range samples and a quadratic fits better. In this page's default example the same sample comes out about 10% higher with a linear fit than with a quadratic. Judge by R² and by how close each standard back-calculates to its nominal value.
What if a sample reads outside the standard curve?+
Don't extrapolate. Above the top standard, dilute the sample further, rerun it and multiply by the new dilution factor. Below the lowest non-zero standard, dilute less or switch to the enhanced protocol (60 °C for 30 minutes, working range 5 to 250 μg/mL). The calculator gives no concentration for out-of-range samples.
How do I account for the dilution factor?+
Enter how many times you diluted the sample before the assay. For example, 10 μL of lysate plus 40 μL of diluent is a 5-fold dilution; if the curve reads 591 μg/mL, the original sample is 591 × 5 = 2955 μg/mL, about 2.96 mg/mL. Leave it blank for undiluted samples.
What interferes with the BCA assay?+
Reducing agents (DTT, β-mercaptoethanol) and chelators (EDTA) interfere with BCA color development; Thermo's TR0008 note reports interference from samples containing 5 mM DTT. Options: use a reducing-agent-compatible BCA kit, remove the interfering substances by TCA or acetone precipitation, or dialyze or dilute. Prepare the standards in the same lysis buffer as your samples where possible.
Why must standards and samples be read at the same time?+
The BCA reaction has no true endpoint: the color keeps developing after incubation. Standards must be on the same plate as the samples, incubated together (37 °C for 30 minutes) and read at 562 nm together as soon as possible. Reading the standards first and the samples later shifts the curve.
My BCA was accurate, so why are my Western blot loading controls uneven?+
Quantification is only the first step. Pipetting error when loading, precipitate in samples that weren't spun after boiling, uneven transfer and saturated loading-control signal all make bands uneven. High-abundance controls such as GAPDH or β-actin can already be outside the linear range at typical loads, so run a loading series to confirm the signal is linear, or normalize to Ponceau or total protein stain.

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