Microsoft PowerPoint - Chapter 3-2

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1 Chapter 3 단백질의탐구및단백체학

(3) 겔거르기 (Gel filtration) chromatography: 크기에의한분리 - Resin: 다공성구슬 = Sephadex, Sepharose, Biogel = Bead size: 100 mm - 원리 : 분배 (partition) chromatograph = 분배 : 용질이이동상 (mobile phase) 과고정상

2 (3) 겔거르기 (Gel filtration) chromatography: 크기에의한분리 - Resin: 다공성구슬 = Sephadex, Sepharose, Biogel = Bead size: 100 mm - 원리 : 분배 (partition) chromatograph = 분배 : 용질이이동상 (mobile phase) 과고정상 (stationary phase) 에분배되는정도 = 이동상 : 완충용액 = 고정상 : resin = 배제한계 (Exclusion limit): 레진의구멍의크기에의해결정 - 배제한계보다큰물질 : Resin 사이의공극을통해이동 - 배제한계보다작은물질 : Resin 의구멍을통해이동 - Void volume: - Gel volume: - Elution volume:

4 (4) 이온교환 (ion-exchange) chromatography: 전하의차이를이용 - Resin: 전하를띈 = 음이온교환 (Anion): DEAE-cellulose = 양이온 (Cation): CM-cellulose - 분자의분리는분자의전하에따라분리된다 - 용출은 buffer 의염농도를높인다

5 (5) 친화 (Affinity) chromatography: 단백질결합의특이성을활용 - Resin: antibody-linked, Substrate-linked - 장점 : 한번에원하는단백질의분리가가능함 - 용출 : = Antibody: low ph (ph 3.0) = Substrate-Enzyme: substrate

6 (6) 고압액체크로마토드래피 (High pressure liquid chromatography;hplc) - 용매의압력을높여액체크로마토그래피의성능을향상시킴 - 시간을절약할수있고, 분해능이향상됨 - 일반액체 chromatography 의단점을보완 = low resolution: = Time consuming:

7 4) 전기이동 ( 영동 ) 법에의한단백질의분리및확인 - 단백질분리효율을측정하는방법 = To check increase of specific activity: assay = To check decrease of protein contaminant: electrophoresis (1) Gel electrophoresis - 전기영동 : 전하를띈물질이전기장안에서움직이는현상 v = Ez/f v: 이동속도 E: 전기장의세기 z: 분자의알짜전하 f: 마찰계수 f = 6πηr π: pi η: 용매의점도 r: 분자의반지름

8 - gel electrophoresis 의매질 (matrix) = DNA/RNA: Agarose gel = Protein : polyacrylamide gel - 전기영동시단백질의분리요인 : = Size = Charge = Shape - Polyacrylamide gel = Native gel electrophoresis: 단백질의알짜전하와모양에의해분리 = Denatured gel electrophoresis: 크기 Soduim dodecyl sulfate (SDS) beta-mercaptoethanol = 단백질검출 Autoradiogram: Western Coomassie blue staining SDS

10 (2) 등전점전기영동 (Isoelectric focusing) - 등전점 (isoelectric point; pi): t 단백질의알짜전하가 0 이되는 ph. - 단백질의 pi 는이온성아미노산에의해결정된다. - 등전점에서단백질은전기장안에서움직이지않는다. - polyacrylamide gel 안에서 ph gradient 를만드는방법. = polyampholytes

11 (3) 2 차원전기영동 (Two-dimensional electrophoresis) - 등전점전기영동법과 SDS- 전기영동법을합쳐단백질을분리하는기술 = 1 차원전기영동 : 등전점전기영동 = 2 차원전기영동 : SDS-PAGE - 이기술은단백질체분석에이용됨.

12 Application of 2D-gel electrophoresis on the proteomics analysis

13 5) 단백질정제계획을정량적으로검토할수있다. - 각정제단계의효율을측정하는방법 = 비활성 (specific activity) 의증가를측정하는방법 : assay = 단백질순도를측정하는방법 : electrophoresis (1) To check increase of specific activity (2) To check protein content

14 6) 초원심분리는생체분자를분리하고그분자량을결정할수있다. - 원심분리 : = 생체물질의침강은질량과밀도에의하여분리됨 mass and density. = 침강계수 : 원심력이작용하는장소에서입자들이움직이는속도. s = m(1-υρ)/f m: mass υ: particle specific volume ρ: density of medium f: frictional coefficient = Svedberg unit (S): ribosome small subunit (40 S), large subunit (60 S)

15 - 농도구배원심분리 : = 자연상태의생체분자들의분자량을측정할수있게함. cf) SDS-PAGE 법은변성된단백질의분자량을측정할수있음.

16 3.3 면역학은단백질을연구하는중요한기법들을제공한다. 1. 특정단백질에대한항체를만들수있다 - Antibody: Immunoglobulin G (IgG) - Antigen: = Epitope (antigenic determinant) - 항체의생산 = Immunization: inject antigen into rabbit two times (boosting) = Antiserum: = Polyclonal antibody:

17 Antigen-Antibody reaction

18 2. 단일클론항체 (Monoclonal antibody) 생산 - Single epitope binding antibody

19 3. 단백질검출은 ELISA 기술을이용하여가능하다. - 샘플속에 ng 단백질을검출할수있음 = AIDS virus 단백질검출 = 혈액중 Insulin 측정 - ELISA (Enzyme Linked Immunosorbent Assay) = 간접법 : 특정항원에특이적인항체의존재유무를측정함 = Sandwich ELISA: 항원을직접측정할수있음.

20 4. Western blotting 기술은 gel electrophoresis 에의해분리된단백질을측정할수있다. - SDS-PAGE: 단백질크기에의한분리 - 단백질의검출은특이항체 (primary Ab) 에의해결정되고, 그항체는 anti-igg 항체 (2 nd antibody) 에의해검출됨

21 5. 형광표지물은단백질을눈으로확인할수있게해준다. - 형광물질이붙어있는항체는세포내단백질을검출할수있음 = Fluorescent microscopy = Immunohistochemistry = Confocal microscopy ( 공촛점현미경 ) - Green fluorescent protein (GFP): real time protein location

22 3.5 Mass spectrometry 1. The Mass of a protein can be determined by Mass spectrometry. - Ionization method = Matrix-Assisted Laser Desorption-Ionization (MALDI) = Electrospray ionization (ESI): - Separation of mass (determine the mass of proteins or peptide): = Time of flight (TOF) - Mass spectrometry: MALDI-TOF

3.6 3 차원단백질의구조는 X-ray 결정학과 NMR 분광법으로결정될수있다. 1.

radiation - Detector: Detect the scattered beam.

23 3.6 3 차원단백질의구조는 X-ray 결정학과 NMR 분광법으로결정될수있다. 1. X-ray 결정학은단백질의 3 차구조를밝힐수있음. - Protein crystal: depends on the proteins - Source of X-ray: x-ray generator or synchrotron radiation - Detector: Detect the scattered beam. - Electron density determination Resolution is depended on wave length

24 2. NMR 분광법은용액속에존재하는단백질을검출할수있음. - Calculate the hydrogen atom distribution in molecules.

(Establishment and Management of Proteomics Core Facility)

(Establishment and Management of Proteomics Core Facility) 1 sample running on SDS-PAGE Gel (10well) 163570 10 16357 1D size marker 225500 100 2255 Buffer(20X, 500ml) 119900 20 5995 Staining