195- The Bio-Rad Power Pac 3000 system or similar.
- Separating gel (12 %): mix 1.625 ml H 2 O, 2 ml 30 % acryl-
amide–Bis-acrylamide solution (29.2:0.8 acrylamide–Bis-
acrylamide), 1.3 ml 1.5 M Tris/HCl buffer, pH 8.8, 25 μl
20 % SDS; 50 μl 10 % ammonium persulfate (APS) and 2 μl
tetramethylethylenediamine (TEMED). - Stacking gel (3.9 %): mix 2.64 ml H 2 O, 0.67 ml 30 % acryl-
amide–Bis-acrylamide solution, 0.5 ml 1 M Tris/HCl buffer,
pH 6.8, 20 μl 20 % SDS; 40 μl 10 % APS, 8 μl TEMED, and
20 μl bromophenol blue. - 4× Tris/HCl/SDS solution: 91 g Tris base, 2 g SDS, 500 ml
H 2 O, pH 8.8. - 2× SDS sample loading buffer (100 ml fi nal volume): 25 ml 4×
Tris/HCl/SDS solution, 20 ml glycerol, 4 g SDS, 2 ml
β-Mercaptoethanol (or 3.1 g DTT), 1 mg bromophenol blue,
adjust to 100 ml with H 2 O. - 10× SDS electrophoresis running buffer: 250 mM Tris base,
1.92 M glycine, 1 % SDS. Adjust pH to pH 8.3 when diluting
to 1×. - Gel staining solution: 50 % methanol, 0.05 % Coomassie
Brilliant Blue R-250, 10 % acetic acid. - Gel destaining solution: 5 % methanol, 7 % acetic acid.
- The Bio-Rad Power Pac 3000 system or similar.
- 10× gel transfer buffer: 250 mM Tris base, 1.92 M glycine.
- 1× gel transfer buffer solution: 10 ml 10× gel transfer buffer,
20 ml methanol, pH 8.3–8.4, adjust to 1 L with H 2 O. - Nitrocellulose membrane (Whatman, GE Healthcare Life sci-
ences or similar). - Filter paper, cut to the size of the gel.
- Ponceau S Solution: 0.5 g Ponceau S, 1 ml glacial acetic acid,
adjust to 100 ml with H 2 O. - 1× Tris Buffered Saline supplemented with tween 20 (TBST):
25 mM Tris, 150 mM NaCl, 3 mM KCl, pH 7.5, 0.1 %
Tween 20. - Blocking buffer: 1× TBST, 5 % nonfat dry milk (NFDM).
- Primary antibody: 1/200 anti-Flag antibody or 1/1,000 anti-
Histidine antibody in TBST with 1 % NFDM. - Secondary antibody: 1/2,500 horseradish peroxidase-
conjugated antibody in TBST with 1 % NFDM. - Glass plate.
- ECL Western Blotting Detection Reagent (GE Healthcare).
2.6 Sodium Dodecyl
Sulfate–
Polyacrylamide Gel
Electrophoresis
(SDS-PAGE)
2.7 Western Blotting
Leishmania Metacaspase