فحص البروتين اللوني (طريقة برادفورد)

قياس تركيز البروتين باستخدام ربط صبغة كوماسي بريليانت بلو

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

الهدف

تحديد تركيز البروتين في عينة مجهولة باستخدام فحص برادفورد ومنحنى معايرة BSA.

الخلفية

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.

تحذيرات السلامة

  • 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

معدات الوقاية الشخصية المطلوبة

goggles gloves lab_coat

المواد

  • 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)

المعدات

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

الإجراء

1

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

10 دقيقة
2

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

5 دقيقة
3

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

5 دقيقة Reagent contains acid
4

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

5 دقيقة
5

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

15 دقيقة
6

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

10 دقيقة
7

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

10 دقيقة

النتائج المتوقعة

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.

التنظيف

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

التفاصيل

الفئة
Colorimetry
الصعوبة
Advanced (University)
المدة
60 دقيقة
التكلفة التقديرية
$15,00
الخطوات
7
المواد
4

مزيد من التجارب

التحديد الطيفي للأسبرين 75 min التحديد اللوني للجلوكوز 60 min التحديد اللوني للفوسفات 90 min التحقق من قانون بير-لامبرت 60 min التحليل الطيفي الضوئي لخليط 75 min تحديد الحديد في الماء 75 min تحليل صبغة الطعام بالمطياف الضوئي 60 min

Frequently Asked Questions

What is the objective of فحص البروتين اللوني (طريقة برادفورد)?
تحديد تركيز البروتين في عينة مجهولة باستخدام فحص برادفورد ومنحنى معايرة BSA.
How difficult is فحص البروتين اللوني (طريقة برادفورد)?
This experiment is rated as Advanced (University). It takes approximately 60 minutes to complete.
What safety precautions are needed for فحص البروتين اللوني (طريقة برادفورد)?
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 فحص البروتين اللوني (طريقة برادفورد)?
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 فحص البروتين اللوني (طريقة برادفورد)?
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.