Western Blot Analysis using His-Tag Antibody

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1 Western Blot Analysis using His-Tag Antibody
조교 : 최 혜 정(s338)

2 Today.... Chapter6. protein overexpression in E.coli and purification by affinity chromatography Chapter8. SDS PAGE & Bradford assay Chapter9. Western blot analysis using His-Taq antibody

3 Today.... PAGE gel Transfer to membrane Blocking
Primary Ab binding & Secondary Ab binding Detection

4 Sample(His fusion protein)
IPTG induction > protein overexpression in E.coli Input, FT, wash, binding sample 10% SDS - PAGE gel loading

5 Blotting ? 1. Southern blotting 2. Northern blotting
DNA molecules을 agarose gel로부터 membrane으로 옮긴 후, 특정적인 DNA sequence를 probe로 사용하여 detection해내는 방법 2. Northern blotting Northern blot은 Southern blot과 같은 원리이며, DNA 대신 특정한 RNA를 detection하는 방법이다.

6 Blotting ? 3. Western blotting 단백질만을 찾아내는 방법. -Immunoblotting 라고도 함.
- Antigen-Antibody reaction을 이용하여 여러 proteins 혼합물 중에서 원하는 특정 단백질만을 찾아내는 방법. -Immunoblotting 라고도 함. -원하는 protein에 대한 high-quality antibody가 필요.

7 Experiments Using Antibody
-western blotting   -immunohistochemistry -ELISA (Enzyme-Linked Immunosorbent Assay) -immunoperoxidase cell staining -immunofluorescnce cell staining -immunoprecipitation -flow cytometry

8 Principles of Western blotting
1. Advantages of Western blotting - To test antigenicity of specific proteins - To identify specific proteins - To identify their molecular weights - 소량의 protein도 detection이 가능 2. The basic blotting procedure - Preparation of the antigen sample - Resolution of the sample by gel electrophoresis - Transfer of the separated polypeptides to a membrane support - Blocking nonspecific binding sites on the membrane - Addition of the antibody(primary Ab & secondary Ab) - Detection

9 Principles of Western blotting
SDS-PAGE

10 Principles of Western blotting
Transfer - polyacrylamide gel에서 protein sample을 분리한 후, proteins를 ( gel에서 분리된 양상대로 ) membrane에 옮기는 과정 * Membrane을 사용하는 이유? 1) concentration of protein 2) antibody를 이용한 detection이 수월하다. 3) gel보다 다루기가 손쉽다. 4) staining 및 destaining이 빠르다. 5) 장기간의 보관이 용이하다. * Transfer buffer 1) 20% methanol : membrane에 존재하는 protein의 binding capacity 증가 2) 0.1% SDS – low conc. of SDS in buffer can improve transfer efficiency .

11 Principles of Western blotting
Transfer membrane의 종류 및 특징 Membranes 특징 Nitrocellulose (NC) membrane excellent sensitivity excellent resolution low background Polyvinylidene difluoride (PVDF) membrane* high protein binding capacity physical strength and chemical stability Nylon membrane unacceptably high background

12 Principles of Western blotting
(A) Tank transfer (B) Semidry transfer Confirmation of transfer 1. Prestained markers 2. Blot staining- Ponceau S 3. Gel staining- Coomassie™ blue

13 Principles of Western blotting
Blocking - membrane상에서 antibody와 protein의 non-specific binding을 피하기 위함. Common membrane blocking reagents Reagent Formulation Comments Dried milk 5% non-fat dried milk in PBS or TBS Masks some antigens Milk/Tween-20 5% non-fat dried milk in PBS or TBS, 0.1% Tween 20 Tween-20 0. 1% Tween 20, 0.02% NaN 3 in PBS or TBS Can stain after detection BSA 0.3–3% bovine serum albumin, 0.02% NaN 3 in PBS Lower endogenous cross-reactivity Washing - Western blotting은 여러 가지 연속적인 binding steps을 거침. - non-specific binding을 최소화 해주는 과정.

14 Principles of Western blotting
Addition of Primary antibody - primary antibody는 membrane에 붙어있는 target antigen protein을 인식 - antigen 특정 부위에 binding 함 # appropriate dilution이 중요 - Mixture에서 우리가 원하는 protein을 high specificity로 detection - non-specific background는 최소화 하는 범위

15 Principles of Western blotting
Addition of Secondary antibody - 가장 일반적으로 primary antibody을 detection 할 수 있는 system - original primary antibody에 대한 species specific하게 준비 Ex> goat anti-rabbit secondary Antibody for detection of a rabbit primary Antibody - 가장 일반적으로 horseradish peroxidase (HRP) 혹은 alkaline phosphatase (AP)와 같은 reporter enzyme을 가지고 있다 # 적당한 enzyme substrate을 반응 BCIP/NBT Western blotting ECL western blotting

16 Principles of Western blotting
Detection method Commonly used chromogenic and chemiluminescent detection systems System Substrate1 Product Chromogenic HRP DAB brown precipitate, addition of Ni or Co ions increases sensitivity TMB blue precipitate 4CN blue/black precipitate AP BCIP/NBT black/purple precipitate Chemiluminescent (ECL, ECL Plus) Luminol light, glow or flash Dioxetane-phosphate light, glow 1 Abbreviations: ....DAB: 3,3'-diaminobenzidine ....4CN: 4-chloro-1-naphtol ....BCIP: 5-bromo-4-chloro-3-indolyl phosphate ....NBT: nitroblue tetrazolium ....TMB: 3,3'5,5' tetramethylbenzidine

17 Materials 1. Electrophoresis 2. Transfer 3. Blocking
- SDS-PAGE gel (10%) - 1X Tris-glycerine electrophoresis buffer (25 mM Tris-base, 250 mM glycine, 0.1% (w/v) SDS) 2. Transfer - Transfer buffer (48 mM Tris-base, 39 mM glycine, 20% Methanol) - Nitrocellulose membrane 3. Blocking - Blocking buffer (5% (w/v) non-fat dried milk, 0.05% (w/v) sodium azide in TBST) 4. Binding of 1st Antibody - Primary antibody in 1% non-fat dried milk (1:500 dilution) 5. Binding of 2nd Antibody - Secondary antibody in 1% non-fat dried milk (1:2000 dilution) 6. Washing - TBST (150 mM NaCl, 50 mM Tris-Cl (pH 8.0), 0.1% Tween 20)

18 Methods 1. SDS-polyacrylamide gel 만들기 2. Sample 준비
3. Gel electrophoresis -Gel caster에서 gel을 떼어낸 다음 tank에 장착하고 tank buffer를 채운다. -Sample 을 loading한 후 dye가 바닥에 올 때까지 running한다. 4. Transfer -membrane, Wattman paper(4장 준비)를 sponge 크기에 맞게 미리 자른 후 transfer buffer에 담가둔다. -Transfer sandwich 만들기  (white) sponge/paper/membrane/gel/paper/sponge (black) -transfer tank 에 장착하고 transfer한다. (90V, 300 mA, 40 mins) Gel/Membrane Sandwich

19 Methods 5. Blocking (30 min/ RT) 6. 1st Ab binding/TBST (30 min/RT)
5% non-fat milk in TBST 5ml에 membrane을 담가두고 rocker에 둔다. 6. 1st Ab binding/TBST (30 min/RT) washing solution을 버리고 1% non-fat milk in TBST 5ml 에 mouse anti-His Ab 10ul (1:500)를 부운 후에 rocker에 30분간 둔다. 7. Washing primary Ab를 버리고 Rocker위에서 TBST 로 5분간 3번 씻어준다. 8. 2nd Ab binding/TBST (20 min/RT) washing solution을 버리고 1% non-fat milk in TBST 5ml 에 goat anti-mouse IgG-AP conjugated Ab 2.5ul (1;2000) 를 부은 후에 rocker에 20분간 둔다. 9. Washing secondary Ab를 버리고 Rocker위에서 TBST 로 5분간 3번 씻어준다. 10. AP staining NBT/BCIP solution 10ml을 바로 membrane에 붓고 band가 나타날 때까지 rocking 해준다. 12. 확인 발색되는 band를 확인한 후에는 5ml TBST로 씻어준다.

20 Result CBB BCIP/MBT Western blotting ECL Western blotting M; marker
FT W P 200 Result 116 97.4 66 45 M; marker I; input (crude extract) FT; flow through W; washing P; purified 31 21.5 CBB M I FT W P I FT W P 250 148 98 64 50 36 22 16 BCIP/MBT Western blotting ECL Western blotting

21 Markers

22 Result ??? WB using His Ab 130kDa 100kDa 70kDa 50kDa CBB staining

23 Discussion & Further Study
Antibody를 이용한 실험 – 원리 및 이용법 정리 2. Monoclonal Ab vs. Polyclonal Ab 차이점 정리 실험 목적, discussion, further study  handwritting!! 11/27(금) pm6:00 s338 제출하세요!!