Колориметрический анализ белков (метод Брэдфорда)

Количественное определение концентрации белка с использованием кумасси ярко-синего

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

Цель

Определить концентрацию белка в неизвестной пробе методом Брэдфорда с использованием калибровочной кривой по бычьему сывороточному альбумину (БСА).

Предпосылки

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.

Предупреждения по безопасности

  • 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

Требуемые СИЗ

goggles gloves lab_coat

Материалы

  • 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)

Оборудование

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

Процедура

1

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

10 мин
2

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

5 мин
3

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

5 мин Reagent contains acid
4

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

5 мин
5

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

15 мин
6

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

10 мин
7

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

10 мин

Ожидаемые результаты

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.

Уборка

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

Детали

Категория
Colorimetry
Сложность
Advanced (University)
Продолжительность
60 мин
Прим. стоимость
$15,00
Шаги
7
Материалы
4

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Frequently Asked Questions

What is the objective of Колориметрический анализ белков (метод Брэдфорда)?
Определить концентрацию белка в неизвестной пробе методом Брэдфорда с использованием калибровочной кривой по бычьему сывороточному альбумину (БСА).
How difficult is Колориметрический анализ белков (метод Брэдфорда)?
This experiment is rated as Advanced (University). It takes approximately 60 minutes to complete.
What safety precautions are needed for Колориметрический анализ белков (метод Брэдфорда)?
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 Колориметрический анализ белков (метод Брэдфорда)?
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 Колориметрический анализ белков (метод Брэдфорда)?
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.