Western Blot Analysis

using
His-Tag Antibody

drh. Dyah Ayu Oktavianie, M.Biotech

Today....

• PAGE gel

• Transfer to membrane

• Blocking

• Primary Ab binding & Secondary Ab binding

• Detection
 Blotting ?

1. Southern blotting
DNA molecules, agarose gel, membrane,
DNA sequence, probe detection

2. Northern blotting
Northern blot, Southern blot,
DNA, RNA를 detection.
 Blotting ?

3. Western blotting
- Antigen-Antibody reaction, proteins

-Immunoblotting
protein, high-quality antibody.
 Experiments Using Antibody

-western blotting
-immunohistochemistry
-ELISA (Enzyme-Linked Immunosorbent Assay)
-immunoperoxidase cell staining
-immunofluorescnce cell staining
-immunoprecipitation
-flow cytometry
 Principles of Western blotting

1. Advantages of Western blotting

- To test antigenicity of specific proteins
- To identify specific proteins
- To identify their molecular weights
- protein detection

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
 Principles of Western blotting

SDS-PAGE
 Principles of Western blotting
Transfer
- polyacrylamide gel, protein sample.

* Membrane
1) concentration of protein
2) antibody detection
3) gel
4) staining, destaining

* Transfer buffer
1) 20% methanol :
2) 0.1% SDS – low conc. of SDS in buffer can improve transfer efficiency .
 Principles of Western blotting

Transfer membrane

Membranes Advantages

Nitrocellulose (NC) membrane excellent sensitivity

excellent resolution
low background

Polyvinylidene difluoride (PVDF) high protein binding capacity

membrane* physical strength and chemical stability

Nylon membrane unacceptably high background

 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
 Principles of Western blotting

Blocking
- membrane antibody, protein, non-specific binding.

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 Masks some antigens

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
 Principles of Western blotting

Addition of Primary antibody

- primary antibody, membrane, target antigen protein
- antigen binding

# appropriate dilution
- Mixture protein
high specificity detection
- non-specific background
 Principles of Western blotting

Addition of Secondary antibody

- primary antibody detection system
- original primary antibody, species specific
Ex> goat anti-rabbit secondary Antibody for detection of a rabbit primary Antibody
- horseradish peroxidase (HRP), alkaline phosphatase (AP)
reporter enzyme

BCIP/NBT Western blotting ECL western blotting

 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)
HRP Luminol light, glow or flash
AP Dioxetane- light, glow
phosphate
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
 Materials
1. Electrophoresis
- 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)
 Methods

1. SDS-polyacrylamide gel
2. Sample
3. Gel electrophoresis
4. Transfer

Gel/Membrane Sandwich
 Methods

5. Blocking (30 min/ RT)

5% non-fat milk in TBST 5ml membrane을 담가두고 rocker에 둔다.
6. 1st Ab binding/TBST (30 min/RT)

7. Washing
8. 2nd Ab binding/TBST (20 min/RT)

9. Washing
10. AP staining
 M I FT W P
200
116
97.4
Result
66

45
M; marker
31
I; input (crude extract)
FT; flow through
W; washing
21.5 P; purified

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

Markers
 Result

130kDa ???
100kDa WB using His Ab
70kDa
50kDa

CBB staining