Uji Protein Kolorimetrik (Metode Bradford)

Mengkuantifikasi konsentrasi protein menggunakan pengikatan pewarna Coomassie Brilliant Blue

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

Tujuan

Tentukan konsentrasi protein sampel yang tidak diketahui menggunakan uji Bradford dan kurva kalibrasi BSA.

Latar Belakang

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.

Peringatan Keselamatan

  • 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

APD yang Diperlukan

goggles gloves lab_coat

Bahan

  • 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)

Peralatan

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

Prosedur

1

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

10 menit
2

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

5 menit
3

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

5 menit Reagent contains acid
4

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

5 menit
5

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

15 menit
6

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

10 menit
7

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

10 menit

Hasil yang Diharapkan

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.

Pembersihan

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

Detail

Kategori
Colorimetry
Tingkat Kesulitan
Advanced (University)
Durasi
60 menit
Perkiraan Biaya
$15,00
Langkah
7
Bahan
4

Eksperimen Lainnya

Analisis Pewarna Makanan dengan Spektrofotometri 60 min Analisis Spektrofotometrik Campuran 75 min Penentuan Aspirin Spektrofotometrik 75 min Penentuan Besi dalam Air 75 min Penentuan Fosfat Kolorimetrik 90 min Penentuan Glukosa Kolorimetrik 60 min Verifikasi Hukum Beer-Lambert 60 min

Frequently Asked Questions

What is the objective of Uji Protein Kolorimetrik (Metode Bradford)?
Tentukan konsentrasi protein sampel yang tidak diketahui menggunakan uji Bradford dan kurva kalibrasi BSA.
How difficult is Uji Protein Kolorimetrik (Metode Bradford)?
This experiment is rated as Advanced (University). It takes approximately 60 minutes to complete.
What safety precautions are needed for Uji Protein Kolorimetrik (Metode 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 Uji Protein Kolorimetrik (Metode 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 Uji Protein Kolorimetrik (Metode 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.