SDS-PAGE
Preparation
1. Set casting frames on casting stands
2. Insert an appropriate comb between the glass plates and mark (with a pen) 1.5cm below the lower limit of the comb. Then,
remove the comb
3. Prepare the resolving gel solution in a small beaker
4. Stirr the solution gently
5. Pipet an appropriate amount of resolution gel solution into the gap between glass plates until the pen mark
6. Pipet isopropanol (or 0.1%SDS) on the top of the sandwich until overflow (to make an horizontal top edge of the resolving gel)
7. Wait until resolving gel is completely polymerized (monitor the process by the solution left in the beaker)
8. After polymerization, discard the water and pipet the stacking gel until overflow
9. Insert the comb to form protein wells and wait until it polymerizes. Then, remove the comb
Performing Electrophoresis
1. Prepare the running buffer and fill electrophoresis lower chamber
2. Place your casted gels into the electrophoresis tank
3. Mix your protein sample with sample buffer (2xLaemmli buffer) (1:1)
4. Heat them for 5-10min
5. Load samples into wells
6. Load protein markers (ladder) into the first (very left) well
7. Complete the upper electrophoresis chamber with the running buffer
8. Cover the top and connect the anodes
9. Set an appropriate voltage and run electrophoresis (usually 1hour for 150V, 12% resolving gels)
10. After electrophoresis is done, disassemble electrophoresis tank and gel sandwiches.
11. Coomassie stain overnight. Then, destain in dH2O to visually protein bands.
Handcasting recipes for polyacrylamide gels
Volume needed: 4.2mL (for 0.75mm combs), 5.6mL (for 1mm combs) or 8.4mL (for 1.5mm combs)
Stacking and Resolving gels
Compound
30% Acrylamide/0.8%Bis
0.5M Tris HCL, pH6.8
1.5M Tris HCl, pH8.8
10%SDS
dH2O
TEMED
10%APS
Stacking gel
4%
1.98mL
3.78mL
150uL
9mL
15uL
75uL
Resolving gel
12%
6mL
3.75mL
150uL
5.03mL
7.5uL
75uL
Total volume
15mL
15mL
2x Laemmli Buffer
65.8mM Tris-HCl, pH 6.8; 2.1%SDS; 26.3% (w/v) glycerol; 0.01% bromophenol blue
10x Running buffer (use 1x)
250mM Tris; 1.92M glysine; 1% SDS