Phân Tích Protein Bằng Phép Đo Màu (Phương Pháp Bradford)

Định lượng nồng độ protein bằng liên kết thuốc nhuộm Xanh Coomassie Brilliant

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

Mục tiêu

Xác định nồng độ protein của mẫu chưa biết bằng phân tích Bradford và đường chuẩn BSA.

Bối cảnh

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.

Cảnh báo an toàn

  • 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

PPE cần thiết

goggles gloves lab_coat

Vật liệu

  • 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)

Thiết bị

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

Quy trình

1

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

10 phút
2

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

5 phút
3

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

5 phút Reagent contains acid
4

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

5 phút
5

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

15 phút
6

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

10 phút
7

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

10 phút

Kết quả dự kiến

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.

Dọn dẹp

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

Chi tiết

Danh mục
Colorimetry
Độ khó
Advanced (University)
Thời gian
60 phút
Chi phí ước tính
$15,00
Số bước
7
Vật liệu
4

Thí nghiệm khác

Phân Tích Phẩm Màu Thực Phẩm Bằng Quang Phổ 60 min Phân Tích Quang Phổ Hỗn Hợp 75 min Xác Định Aspirin Bằng Quang Phổ 75 min Xác Định Glucose Bằng Phép Đo Màu 60 min Xác Định Photphat Bằng Phép Đo Màu 90 min Xác Định Sắt Trong Nước 75 min Xác Minh Định Luật Beer-Lambert 60 min

Frequently Asked Questions

What is the objective of Phân Tích Protein Bằng Phép Đo Màu (Phương Pháp Bradford)?
Xác định nồng độ protein của mẫu chưa biết bằng phân tích Bradford và đường chuẩn BSA.
How difficult is Phân Tích Protein Bằng Phép Đo Màu (Phương Pháp Bradford)?
This experiment is rated as Advanced (University). It takes approximately 60 minutes to complete.
What safety precautions are needed for Phân Tích Protein Bằng Phép Đo Màu (Phương Pháp 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 Phân Tích Protein Bằng Phép Đo Màu (Phương Pháp 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 Phân Tích Protein Bằng Phép Đo Màu (Phương Pháp 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.