CodonTableCodonTable
Protein Biochemistry
BCA & Bradford Standard Curve Interpolation
Direct UV A280 Beer-Lambert

Protein Concentration Calculator (BCA, Bradford & UV A280)

Determine protein concentration by interpolation between measured BCA or Bradford standards, with a separate linearity diagnostic, or calculate concentration using direct UV A280 Beer-Lambert spectrophotometry.

Standard Curve Analysis

Example values are preloaded. Replace them with standards measured under the same assay conditions as your sample.
#Std Conc (µg/mL)Absorbance (OD)Action
1
2
3
4
5
6
7
8
8 points defined

Unknown Protein Sample Reading

Standard Curve Results

1.57002200Concentration (µg/mL)
Linear diagnostic equation:y = 0.000643 · x + 0.0879
Slope (m):6.4335e-4 OD·mL/µg
Intercept (b):0.0879 OD
Linear-fit diagnostic (R²):0.9998

Sample concentration uses piecewise linear interpolation between adjacent measured standards. R² describes a separate linear fit and is a curve-linearity diagnostic.

Calculated Protein Concentration (DF = 5×)
3.050 mg/mL
= 3049.7 µg/mL

Protein Concentration Calculator Principles: BCA, Bradford & UV A280

Accurate quantification of protein concentration is fundamental to molecular biology, antibody characterization, enzymatic assays, and Western blotting. Three principal analytical methods dominate standard laboratory practice:

BCA Assay (562 nm)

Cu²⁺ is reduced to Cu⁺ by peptide bonds and cysteine, cystine, tryptophan, and tyrosine residues in alkaline medium, then chelated by bicinchoninic acid to produce a purple complex. Detergent-compatible (up to 5% SDS, Triton X-100).

Bradford Assay (595 nm)

Coomassie Brilliant Blue G-250 binds to basic (primarily arginine) and aromatic amino acid residues, shifting its absorbance maximum from 465 nm to 595 nm. Highly compatible with reducing agents (DTT, β-ME).

Direct UV A280

Direct spectrophotometric quantification based on intrinsic absorption by Trp, Tyr, and disulfide cystine bonds. Fast, non-destructive, requires no dye or incubation, but vulnerable to nucleic acid cross-contamination.

Protein Quantification Chemical Compatibility: Detergents & Reducing Agents

Interfering ReagentBCA AssayBradford AssayDirect UV A280
Detergents (SDS, Triton X-100, NP-40)Compatible (up to 5%)Incompatible (> 0.1% precipitates)Compatible
Reducing Agents (DTT, β-ME, TCEP)Incompatible (reduces Cu²⁺ directly)Compatible (up to 5 mM DTT)Compatible (with buffer blank)
Nucleic Acids (DNA / RNA)No interferenceMinimal interferenceSevere (A260 spillover)

Protein Concentration Calculator Worked Lab Example: BCA Standard Curve

Scenario: Cell Lysate Protein Determination via BCA Assay

A RIPA cell lysate is diluted 1:5 (DF = 5). Its measured A₅₆₂ is 0.485; the adjacent BSA standards are 500 µg/mL at A = 0.412 and 750 µg/mL at A = 0.578.

  • 1. Bracket the measured absorbance: 0.412 < 0.485 < 0.578
  • 2. Diluted concentration (linear interpolation): 500 + ((0.485 − 0.412) / (0.578 − 0.412)) × 250 = 609.9 µg/mL
  • 3. Original lysate concentration: 609.9 µg/mL × 5 (DF) = 3049.7 µg/mL ≈ 3.05 mg/mL
  • 4. Western blot loading: For 30 µg protein per lane, pipette 30 / 3.05 ≈ 9.84 µL lysate.

Frequently Asked Questions (FAQ)

When should I choose BCA over Bradford assay?

Choose BCA if your sample contains detergents like SDS, Triton X-100, or NP-40 (common in membrane protein and RIPA lysates). Choose Bradford if your buffer contains reducing agents like DTT or β-mercaptoethanol.

How should I interpret R² for a protein standard curve?

R² describes a linear fit and is only a diagnostic; it does not by itself establish that a curve or assay range is suitable. Inspect the assay protocol, residuals, replicate variation, and signal range. This calculator estimates sample concentration by interpolation between adjacent measured standards, separately from the displayed linear fit.

What causes DNA contamination to distort A280 readings?

Nucleic acids have an extinction coefficient at 260 nm that is ~30–50 times higher than proteins at 280 nm. Even 5% DNA contamination can double the apparent A280 reading.

Why use A320 background correction in UV spectrophotometry?

Proteins do not absorb at 320 nm. Any positive signal at A320 represents optical light scattering from insoluble protein aggregates or dust. Subtracting A320 removes turbidity artifacts.

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