Ensaio Colorimétrico de Proteínas (Método Bradford)

Quantificação da concentração de proteínas usando a ligação do corante Azul Brilhante de Coomassie

Colorimetry Advanced (University) 60 min ~$15,00

Objetivo

Determinar a concentração de proteínas de uma amostra desconhecida usando o ensaio Bradford e uma curva de calibração de BSA.

Contexto

The Bradford assay is one of the most widely used methods for protein quantification in biochemistry laboratories. Coomassie Brilliant Blue G-250 dye binds to protein molecules, shifting its absorption maximum from 465 nm (unbound, red/brown) to 595 nm (bound, blue). The increase in absorbance at 595 nm is proportional to the amount of protein present. The method is fast, sensitive (1–25 μg/mL range), and relatively free from interference by most common reagents.

Avisos de segurança

  • Bradford reagent contains phosphoric acid and methanol
  • Coomassie dye stains everything it contacts
  • Wear gloves to protect from dye and acid
  • Use disposable cuvettes to avoid staining

EPI necessário

goggles gloves lab_coat

Materiais

  • Bradford reagent (Coomassie Blue G-250) (100 mL)
    Commercial or freshly prepared
  • BSA standard (bovine serum albumin) (10 mL)
    1 mg/mL stock
  • Unknown protein sample (5 mL)
  • Distilled water (200 mL)

Equipamentos

Spectrophotometer Cuvettes (plastic, disposable recommended) Micropipettes (20–1000 μL) Test tubes (8) Vortex mixer

Procedimento

1

Prepare BSA standards: 0, 2, 5, 10, 15, 20, 25 μg/mL by diluting the 1 mg/mL stock in water.

10 min
2

Add 100 μL of each standard and unknown sample to separate test tubes.

5 min
3

Add 5 mL of Bradford reagent to each tube. Mix by gentle vortexing or inversion. Do not create bubbles.

5 min Reagent contains acid
4

Incubate for 5 minutes at room temperature (do not exceed 60 minutes as color will begin to fade).

5 min
5

Measure absorbance at 595 nm. Zero with the 0 μg/mL blank.

15 min
6

Plot the calibration curve (absorbance vs. BSA concentration). Note that the Bradford assay curve is slightly nonlinear at higher concentrations.

10 min
7

Determine the protein concentration of the unknown from the calibration curve. Account for any dilutions made.

10 min

Resultados esperados

The calibration curve shows absorbance increasing with BSA concentration, with slight deviation from linearity above 15 μg/mL. The color change from brown/red to blue is clearly visible. The unknown protein concentration should be determinable within the calibration range.

Limpeza

Dispose of Bradford reagent waste in designated containers. Use disposable cuvettes if possible. Clean any spills immediately as the dye stains permanently.

Detalhes

Categoria
Colorimetry
Dificuldade
Advanced (University)
Duração
60 min
Custo estimado
$15,00
Passos
7
Materiais
4

Mais experimentos

Análise de Corantes Alimentares por Espectrofotometria 60 min Análise Espectrofotométrica de uma Mistura 75 min Determinação Colorimétrica de Fosfato 90 min Determinação Colorimétrica de Glicose 60 min Determinação de Ferro na Água 75 min Determinação Espectrofotométrica de Aspirina 75 min Verificação da Lei de Beer-Lambert 60 min

Frequently Asked Questions

What is the objective of Ensaio Colorimétrico de Proteínas (Método Bradford)?
Determinar a concentração de proteínas de uma amostra desconhecida usando o ensaio Bradford e uma curva de calibração de BSA.
How difficult is Ensaio Colorimétrico de Proteínas (Método Bradford)?
This experiment is rated as Advanced (University). It takes approximately 60 minutes to complete.
What safety precautions are needed for Ensaio Colorimétrico de Proteínas (Método Bradford)?
Key safety precautions include: Bradford reagent contains phosphoric acid and methanol; Coomassie dye stains everything it contacts; Wear gloves to protect from dye and acid.
What materials are needed for Ensaio Colorimétrico de Proteínas (Método Bradford)?
The main materials required are: Bradford reagent (Coomassie Blue G-250), BSA standard (bovine serum albumin), Unknown protein sample, Distilled water.
What results should I expect from Ensaio Colorimétrico de Proteínas (Método Bradford)?
The calibration curve shows absorbance increasing with BSA concentration, with slight deviation from linearity above 15 μg/mL. The color change from brown/red to blue is clearly visible. The unknown protein concentration should be determinable within the calibration range.