Team:Marburg/Protocols/Protocol30
SDS-Page
Preparation of the SDS-Gel
- prepare the separating gel (for 2 gels):
Acrylamide percentage 12% H2O 3.2 ml Acrylamide/Bis-acrylamide(30%/0.8% w/v) 4 ml 1.5M Tris(pH=8.8) 2.6 ml 10% (w/v)SDS 0.1 ml 10% (w/v) ammonium persulfate (APS) 100 μl TEMED 10 μl - fill the gel into the casting frames
- cover gel with isopropanol
- wait for 20-30min to let it gelate
- prepare the stacking gel (for two gels):
Acrylamide percentage 5% H2O 2.975 ml 0.5 M Tris-HCl, pH 6.8 1.25 ml 10% (w/v) SDS 0.05 Acrylamide/Bis-acrylamide(30%/0.8% w/v) 0.67 ml 10% (w/v) ammonium persulfate (APS) 0.05 ml TEMED 0.005 ml - discard isopropanol and fill in the stacking gel
- insert the comb without trapping air under the teeth
- wait for 20-30min to let it gelate
- set the cell into the cell buffer dam, take out the comb and pour the running buffer into the chamber
Preparation of Samples
- mix samples (OD = 1.0) with 100 μl sample buffer (1x):
- denaturate samples at 95°C for 10 minutes
- load 10 μl sample-solution into wells
