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Biochemistry

Bradford protein assay with a BSA calibration curve

A 96-well Bradford assay: BSA standards by serial dilution, blanks and triplicates, a plate layout, absorbance at 595 nm and an unknown read off the calibration curve.

Learning objective

Build a BSA calibration curve on a 96-well plate and determine an unknown protein concentration from its absorbance at 595 nm.

Safety and hazards

  • Bradford reagent contains phosphoric acid and methanol: corrosive and toxic (H226, H302, H314). Wear gloves and eye protection.
  • Coomassie dye stains skin, clothing and plastics permanently.
  • Dispose of reagent and plates as chemical waste, not down the sink.

Reagents and materials

  • BSA stock — 1 mL (2.00 mg/mL)
  • Bradford reagent (1×) — 25 mL
  • Phosphate-buffered saline (PBS) — 10 mLdiluent and blank
  • Unknown protein sample — 0.5 mL
  • Clear flat-bottom 96-well plate
  • Plate reader with a 595 nm filter

Procedure

1
Plate dilution

Prepare seven BSA standards by 2-fold serial dilution in PBS, from 2000 µg/mL down to 31.25 µg/mL, plus a PBS-only blank.

Dilution series:
2-fold · 6 steps · down to 31.25 µg/mL

Serial dilution calculator

2000 → 1000 → 500 → 250 → 125 → 62.5 → 31.25 µg/mL

cₙ = c₀ / foldⁿ

Start concentration
2000 µg/mL
Dilution factor
2-fold
Number of transfers
6 (7 standards)
Transfer / diluent volume
100 µL / 100 µL
2
Plate setup

Pipette 10 µL of each standard, blank and unknown into the 96-well plate in triplicate, following the layout below.

Plate layout:
96-well · 24 wells · 8 groups

Equipment: Multichannel pipette, clear flat-bottom 96-well plate

3
Pipette

Add 200 µL of 1× Bradford reagent to every well used.

  • 200 µL/well Bradford reagent (1×)Reagent
Pipetting transfer:
200 µL · Bradford reagent reservoir → All used wells · ×3
4
Incubation

Incubate the plate at room temperature for 10 minutes, protected from direct light.

Temperature:
20–25 °C
Duration:
10 min
Light condition:
protect from direct light
5
Read plate

Read the absorbance of every well at 595 nm and subtract the mean blank absorbance from all readings.

Wavelength:
595 nm

Equipment: Microplate reader

Example blank-corrected means: 2000 µg/mL 0.960 · 1000 0.612 · 500 0.351 · 250 0.196 · 125 0.108 · 62.5 0.061 · unknown 0.284.

6
Concentration

Fit the calibration curve over the linear range and read the unknown concentration from it.

Calibration curve calculator

Unknown = 378 µg/mL

A = 0.000722·c + 0.0112, R² = 0.998 over 62.5–1000 µg/mL

c = (A − b) / m

Unknown absorbance (blank-corrected)
0.284
Slope m
0.000722 per µg/mL
Intercept b
0.0112
7
Custom measurement

Report the mean and standard deviation of the triplicate unknown wells, and flag any replicate with more than 10 % deviation.

Replicate statistics calculator

378 ± 9 µg/mL (n = 3), RSD 2.4 %

RSD % = (s / x̄) × 100

Replicate absorbances
0.281, 0.284, 0.287

Expected result

  • Blue colour developing within 5 minutes, strongest in the highest standards.
  • Linear calibration with R² ≥ 0.99 between about 62.5 and 1000 µg/mL.
  • Replicate RSD below 5 % with careful pipetting.
  • Saturation above ~1500 µg/mL: dilute the unknown if it reads above the top standard.

Calculators used in this protocol

Serial dilution

f = (C₀ / Cₙ)^(1/n)

Step-by-step transfer and diluent volumes for a dilution series.

Calibration curve

y = m·x + b, x = (y − b) / m

Linear least-squares fit with optional weighting and back-calculated unknowns.

Replicate statistics

RSD % = s / |x̄| × 100

Mean, standard deviation, variance and RSD % for replicate measurements.

Dilution

C1 × V1 = C2 × V2

Solve C1 × V1 = C2 × V2 for any missing variable.

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