lowry assay of protein - cheric...introduction lowry assay 단백질정량분석방법 5 /...
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![Page 1: Lowry assay of protein - CHERIC...Introduction Lowry assay 단백질정량분석방법 5 / 정도의적은양으로도정량이가능. 단백질정제에있어단백질농도측정에널리쓰임](https://reader033.vdocuments.site/reader033/viewer/2022041807/5e553677dac4c55b0e716c90/html5/thumbnails/1.jpg)
Lowry assay of protein
![Page 2: Lowry assay of protein - CHERIC...Introduction Lowry assay 단백질정량분석방법 5 / 정도의적은양으로도정량이가능. 단백질정제에있어단백질농도측정에널리쓰임](https://reader033.vdocuments.site/reader033/viewer/2022041807/5e553677dac4c55b0e716c90/html5/thumbnails/2.jpg)
Introduction Lowry assay 단백질 정량 분석 방법
5㎍/㎖정도의 적은 양으로도 정량이 가능.
단백질 정제에 있어 단백질 농도 측정에 널리 쓰임.
분석가능범위 : 5㎍/㎖~100 ㎍/㎖
소요시간 : 약 40분
실험을 통해 미지의 시료 안에 있는 단백질의 절대량을 구할 수 있다.
![Page 3: Lowry assay of protein - CHERIC...Introduction Lowry assay 단백질정량분석방법 5 / 정도의적은양으로도정량이가능. 단백질정제에있어단백질농도측정에널리쓰임](https://reader033.vdocuments.site/reader033/viewer/2022041807/5e553677dac4c55b0e716c90/html5/thumbnails/3.jpg)
Equipment
Spectrophotometer
Pipets
Voltex mixer
Test tube
Test tube rack
Cuvettes*
![Page 4: Lowry assay of protein - CHERIC...Introduction Lowry assay 단백질정량분석방법 5 / 정도의적은양으로도정량이가능. 단백질정제에있어단백질농도측정에널리쓰임](https://reader033.vdocuments.site/reader033/viewer/2022041807/5e553677dac4c55b0e716c90/html5/thumbnails/4.jpg)
ReagentsTable 1. Reagents for experiment.
Solution 용 질 용 매 Total vol.
A 0.5g CuSO₄5H2O
1g Na3C6H5O7(2H2O)D.W 100ml
B 20g Na2CO3
4g NaOHD.W 1liter
C 1ml Solution A
50ml Solution B . 51ml
D 10ml Folin – Ciocalteu phenol reagent
10ml Distilled water. 20ml
주 의 : Solution A,B 는 상온저장, Solution C,D 는 측정 시 제조
![Page 5: Lowry assay of protein - CHERIC...Introduction Lowry assay 단백질정량분석방법 5 / 정도의적은양으로도정량이가능. 단백질정제에있어단백질농도측정에널리쓰임](https://reader033.vdocuments.site/reader033/viewer/2022041807/5e553677dac4c55b0e716c90/html5/thumbnails/5.jpg)
Fig. 1. Reagents for experiment.
![Page 6: Lowry assay of protein - CHERIC...Introduction Lowry assay 단백질정량분석방법 5 / 정도의적은양으로도정량이가능. 단백질정제에있어단백질농도측정에널리쓰임](https://reader033.vdocuments.site/reader033/viewer/2022041807/5e553677dac4c55b0e716c90/html5/thumbnails/6.jpg)
Experiment
Table 2. Standard conc. (Bovine Serum Albumin)
BSA(㎍)0 4.5 9 15 19.5 30 39 49.5 60 Sample
BSA(150㎍/㎖)
- 0.03 0.06 0.10 0.13 0.2 0.26 0.33 0.4 0.1
D.W 0.5 0.47 0.44 0.4 0.37 0.3 0.24 0.17 0.1 0.4
SolutionC 2.5 2.5
SolutionD 0.25 0.25
A750
㎖
![Page 7: Lowry assay of protein - CHERIC...Introduction Lowry assay 단백질정량분석방법 5 / 정도의적은양으로도정량이가능. 단백질정제에있어단백질농도측정에널리쓰임](https://reader033.vdocuments.site/reader033/viewer/2022041807/5e553677dac4c55b0e716c90/html5/thumbnails/7.jpg)
Fig. 2. 증류수 주입.
![Page 8: Lowry assay of protein - CHERIC...Introduction Lowry assay 단백질정량분석방법 5 / 정도의적은양으로도정량이가능. 단백질정제에있어단백질농도측정에널리쓰임](https://reader033.vdocuments.site/reader033/viewer/2022041807/5e553677dac4c55b0e716c90/html5/thumbnails/8.jpg)
Fig. 3.
BSA 및 sample 주입.
![Page 9: Lowry assay of protein - CHERIC...Introduction Lowry assay 단백질정량분석방법 5 / 정도의적은양으로도정량이가능. 단백질정제에있어단백질농도측정에널리쓰임](https://reader033.vdocuments.site/reader033/viewer/2022041807/5e553677dac4c55b0e716c90/html5/thumbnails/9.jpg)
Fig. 4.
Solution C 주입.
![Page 10: Lowry assay of protein - CHERIC...Introduction Lowry assay 단백질정량분석방법 5 / 정도의적은양으로도정량이가능. 단백질정제에있어단백질농도측정에널리쓰임](https://reader033.vdocuments.site/reader033/viewer/2022041807/5e553677dac4c55b0e716c90/html5/thumbnails/10.jpg)
Fig. 5. Solution C를 넣은 후 교반.
![Page 11: Lowry assay of protein - CHERIC...Introduction Lowry assay 단백질정량분석방법 5 / 정도의적은양으로도정량이가능. 단백질정제에있어단백질농도측정에널리쓰임](https://reader033.vdocuments.site/reader033/viewer/2022041807/5e553677dac4c55b0e716c90/html5/thumbnails/11.jpg)
Fig. 6.
Solution D 주입.
![Page 12: Lowry assay of protein - CHERIC...Introduction Lowry assay 단백질정량분석방법 5 / 정도의적은양으로도정량이가능. 단백질정제에있어단백질농도측정에널리쓰임](https://reader033.vdocuments.site/reader033/viewer/2022041807/5e553677dac4c55b0e716c90/html5/thumbnails/12.jpg)
Fig. 7. Solution D를 넣은 후 교반.
![Page 13: Lowry assay of protein - CHERIC...Introduction Lowry assay 단백질정량분석방법 5 / 정도의적은양으로도정량이가능. 단백질정제에있어단백질농도측정에널리쓰임](https://reader033.vdocuments.site/reader033/viewer/2022041807/5e553677dac4c55b0e716c90/html5/thumbnails/13.jpg)
After 20~30 minutes, read A750.
Fig. 8. 증류수로 cuvette 세척.*
![Page 14: Lowry assay of protein - CHERIC...Introduction Lowry assay 단백질정량분석방법 5 / 정도의적은양으로도정량이가능. 단백질정제에있어단백질농도측정에널리쓰임](https://reader033.vdocuments.site/reader033/viewer/2022041807/5e553677dac4c55b0e716c90/html5/thumbnails/14.jpg)
Fig. 9. sample을 cuvette에 담기.
![Page 15: Lowry assay of protein - CHERIC...Introduction Lowry assay 단백질정량분석방법 5 / 정도의적은양으로도정량이가능. 단백질정제에있어단백질농도측정에널리쓰임](https://reader033.vdocuments.site/reader033/viewer/2022041807/5e553677dac4c55b0e716c90/html5/thumbnails/15.jpg)
Fig. 10. spectrophotometer로 sample OD측정.
![Page 16: Lowry assay of protein - CHERIC...Introduction Lowry assay 단백질정량분석방법 5 / 정도의적은양으로도정량이가능. 단백질정제에있어단백질농도측정에널리쓰임](https://reader033.vdocuments.site/reader033/viewer/2022041807/5e553677dac4c55b0e716c90/html5/thumbnails/16.jpg)
Conc.(㎍/ml) A750 Real A750
0 0.063 0
4.5 0.070 0.007
9 0.125 0.062
30 0.171 0.108
60 0.292 0.229
Table 3. Standard 측정값.
Result
A750에서 측정한 Standard의 값
![Page 17: Lowry assay of protein - CHERIC...Introduction Lowry assay 단백질정량분석방법 5 / 정도의적은양으로도정량이가능. 단백질정제에있어단백질농도측정에널리쓰임](https://reader033.vdocuments.site/reader033/viewer/2022041807/5e553677dac4c55b0e716c90/html5/thumbnails/17.jpg)
Fig. 11. Standard Curve.
Standard curve
y = 0.0037x + 0.004
0
0.05
0.1
0.15
0.2
0.25
0 10 20 30 40 50 60 70Conc.
A750
![Page 18: Lowry assay of protein - CHERIC...Introduction Lowry assay 단백질정량분석방법 5 / 정도의적은양으로도정량이가능. 단백질정제에있어단백질농도측정에널리쓰임](https://reader033.vdocuments.site/reader033/viewer/2022041807/5e553677dac4c55b0e716c90/html5/thumbnails/18.jpg)
Sample의 측정값과 농도 계산
No. A750 Real A750 Conc.
1 0.436 0.373 99.73
2 1.134 1.071 288.38
3 0.63 0.567 152.16
4 0.6 0.537 144.05
5 0.263 0.2 52.97
Table 4. sample의 측정값과 농도.
![Page 19: Lowry assay of protein - CHERIC...Introduction Lowry assay 단백질정량분석방법 5 / 정도의적은양으로도정량이가능. 단백질정제에있어단백질농도측정에널리쓰임](https://reader033.vdocuments.site/reader033/viewer/2022041807/5e553677dac4c55b0e716c90/html5/thumbnails/19.jpg)
No. 단백질의 양(㎍)
1 3241.22
2 9372.30
3 4945.27
4 4681.76
5 1721.62
Table 5. sample의 단백질 양.