비색 단백질 정량(브래드퍼드법)

쿠마시 브릴리언트 블루 염료 결합으로 단백질 농도 정량하기

Colorimetry Advanced (University) 60 분 ~$15.00

목표

브래드퍼드 분석법과 BSA 검량선을 사용하여 미지 시료의 단백질 농도를 결정한다.

배경

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.

안전 경고

  • 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

필수 개인 보호 장비

goggles gloves lab_coat

재료

  • 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)

장비

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

실험 절차

1

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

10 분
2

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

5 분
3

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

5 분 Reagent contains acid
4

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

5 분
5

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

15 분
6

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

10 분
7

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

10 분

예상 결과

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.

정리

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

세부 정보

카테고리
Colorimetry
난이도
Advanced (University)
소요 시간
60 분
예상 비용
$15.00
단계
7
재료
4

더 많은 실험

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Frequently Asked Questions

What is the objective of 비색 단백질 정량(브래드퍼드법)?
브래드퍼드 분석법과 BSA 검량선을 사용하여 미지 시료의 단백질 농도를 결정한다.
How difficult is 비색 단백질 정량(브래드퍼드법)?
This experiment is rated as Advanced (University). It takes approximately 60 minutes to complete.
What safety precautions are needed for 비색 단백질 정량(브래드퍼드법)?
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 비색 단백질 정량(브래드퍼드법)?
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 비색 단백질 정량(브래드퍼드법)?
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.