Professional Documents
Culture Documents
using
His-Tag Antibody
• PAGE gel
• Transfer to membrane
• Blocking
• 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
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
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.
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
# appropriate dilution
- Mixture protein
high specificity detection
- non-specific background
Principles of Western blotting
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)
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
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
130kDa ???
100kDa WB using His Ab
70kDa
50kDa
CBB staining