2.3.2 Sodium Dodecyl
Sulfate-Polyacrylamide Gel
Electrophoresis
(SDS-PAGE)
1. Resolving gel buffer: 1.5 M Tris–HCl pH 8.8, 0.4%
(w/v) SDS.
2. Stacking gel buffer: 0.5 M Tris–HCl pH 6.8, 0.4% (w/v) SDS.
3. Resolving gel solution (8% acrylamide; 15 mL): 4 mL 30%
acrylamide/bis-acrylamide solution, 3.75 mL resolving gel
buffer, 7.25 mL water, 50 μL 10% ammonium persulfate
(APS, prepared fresh), 10 μL N,N,N,N
0 -tetramethyl-ethylenediamine (TEMED).
4. Stacking gel solution (5 mL): 650 μL 30% acrylamide/bisacrylamide solution, 1.25 mL stacking gel buffer, 3.05 mL
water, 25 μL 10% APS, 5 μL TEMED.
5. SDS-PAGE running buffer: 0.025 M Tris, 0.192 M glycine,
0.1% (w/v) SDS.
6. 6Â SDS sample buffer: 0.35 M Tris–HCl pH 6.8, 30% (v/v)
glycerol, 10% (w/v) SDS, 6 M DTT, 0.012% (w/v)
bromophenol blue.
7. Pre-stained protein molecular weight marker.
8. Gel-casting apparatus.
9. Electrophoresis chamber.
10. Power supply
2.3.3 Electro-Transfer
1. Towbin buffer: 0.025 M Tris, 0.192 M glycine, 20% (v/v)
methanol.
2. Electro-blotting apparatus.
3. Power supply.
4. Nitrocellulose membrane, 0.45 μm pore size.
2.3.4 Immunological
Detection
1. Ponceau staining solution: 0.1% (w/v) Ponceau S, 5% (v/v)
acetic acid.
2. Tris-buffered saline (TBS): 50 mM Tris–HCl pH 7.6,
150 mM NaCl.
3. TBS-T: TBS supplemented with 0.1% (v/v) TWEEN 20.
4. Blocking solution: TBS-T supplemented with 5% (w/v) nonfat
dry milk powder.
5. Polyclonal anti-GFP antibody.
6. Horseradish peroxidase (HRP)-conjugated rat monoclonal
anti-HA (3F10) antibody.
7. HRP-conjugated secondary antibody (goat anti-rabbit IgG,
etc.).
8. Clarity Western ECL Substrate.
9. Orbital shaker.
10. Luminescence gel imaging equipment, including software.
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