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BCA Protein Assay Calculator

Calculate protein concentrations from a BCA (bicinchoninic acid) assay: fit the standard curve as a line or a curve, read the unknowns off it and apply dilution factors.

Formula

A562=bc+d(or a second-order polynomial)A_{562} = b c + d\quad(\text{or a second-order polynomial})
c=A−dbc = \dfrac{A - d}{b}
coriginal=c×DFc_{\text{original}} = c \times \mathrm{DF}
cc
protein concentration in the well
b, db,\ d
slope and intercept of the fitted line

How it works

In the BCA assay, protein reduces copper(II) to copper(I) under alkaline conditions, and two bicinchoninic acid molecules chelate each Cu⁺ to give a purple product that absorbs at 562 nm. Peptide bonds and the side chains of cysteine, tyrosine and tryptophan contribute to the reduction.

The response is close to linear over the working range, and the colour keeps developing with time, so standards and samples are incubated together and read at the same time. The tool fits the standards, reports the equation, R² and residuals, and flags unknowns outside the standards.

Worked example

Eight BSA standards from 0 to 2,000 µg/mL (illustrative absorbance values) and two lysates diluted 4- and 2-fold.

  1. The straight-line fit is A = 0.000889 c + 0.0018.
  2. Lysate A (A = 0.350) gives 391.6 µg/mL in the well, times 4 = 1,566 µg/mL.

Lysate A is about 1,570 µg/mL, and lysate B about 1,620 µg/mL (0.720 gives 809.0 in the well, times 2).

These are the values the calculator opens with, so you can check its output against this example.

Assumptions

  • Unknowns and standards react the same way with the reagent.
  • Reducing agents, chelators and other interfering substances are absent or matched in the standards.
  • The standards' absorbances here are illustrative; use your own measured values.

Common mistakes

  • Reading standards and samples at different times or after different incubations.
  • Ignoring reducing agents such as DTT, which raise the signal.
  • Fitting a straight line where the residuals clearly curve.

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Related equipment

Service documentation, failure modes and parts for the instruments this calculation is used with.