165- Pour the separating gel in between the glass plates. Leave
suffi cient space for the stacking gel to be added later (the
length of the comb teeth plus 0.5–1 cm). To provide a sharp
interface, overlay the gel with water during polymerization. - Allow gel to polymerize for 30 min. Remove overlay water
completely. - Prepare the stacking gel. For two gels (one set), combine the
following components:
6.05 mL H 2 O.
2.5 mL Stacking gel buffer, 0.5 M Tris–HCl, pH 6.8.
100 μL 10 % SDS.
1.35 mL Protogel.
45 μL 10 % APS.
25 μL TEMED. - Pour stacking gel solution on top of the polymerized separating
gel. Immediately insert a clean Tefl on comb into the stacking
gel solution. Allow gel to polymerize for 30 min ( see Note 20 ). - Remove the gel comb and assemble electrophoresis system
according to the manufacturer’s instructions. Fill it with 1×
electrode buffer. - Load equal protein amounts from each sample (20–80 μg)
into wells under the electrode buffer with Prot/Elec pipette
tips. Load one well with 3 μL of the prestained SDS-PAGE
protein ladder. In the well next to the last sample 1× sample
buffer may be added to prevent horizontal spreading of sample
proteins ( see Note 21 ). - Attach the electrophoresis apparatus to an electric power sup-
ply. Run the gel at constant 80 V until the bromophenol blue
front is reaching the separating gel. The remaining electropho-
resis can be performed at constant 130 V. - When the bromophenol blue reaches the bottom of the resolv-
ing gel (approximately 2 h), turn off the power supply and
disconnect the electrophoresis system. Remove the stacking
gel and unused lanes. Submerge the remaining gel in transfer
buffer. - Cut nitrocellulose membranes and 3MM papers according to
gel sizes. All the components, including supporting pads,
should be soaked in transfer buffer in advance of assembly of
the electroblot sandwich. Build the sandwich in the gel holder
cassette as follows: foam pad, 3 3MM papers, the gel, nitrocel-
lulose membrane, 3 3MM papers, and foam pad. Precautions
should be taken to avoid air bubbles that would interfere with
an accurate transfer ( see Note 22 ).
3.1.3 Western Blotting
Caspases in Mammalian Cell Cultures