SDS-PAGE
| Materials | |
|---|---|
| Separating Gel (12,5%): | 3.1 mL Polyacrylamide (40%); 4.3 mL
dH2O; 2.5 mL Tris (1.5 M; pH 8.8) Degas! 50 μL Sodium dodecyl sulfate (SDS; 20%); 30 μL Ammonium Persulfate (APS; 10%); 15 μL TEMED |
| Stacking Gel (5%): | 1.25 mL Polyacrylamide (40%); 6.15 mL
dH2O; 2.5 mL Tris (0,5 M; pH 6,8) Degas! 50 μL Sodium dodecyl sulfate (SDS; 20%); 30 μL Ammonium Persulfate (APS; 10%); 15 μL TEMED |
| Marker | Low molecular weight marker (LMW)
High molecular weight marker (HMW) |
| Running Buffer | 25 mM Tris-HCl
192 mL Glycine 0.1% SDS 2 L dH2O |
Procedure
- For gel electrophoresis, first a separating gel (8% - 10% - 12.5% or 15% polyacrylamide) is poured and gently covered with dH2O. The remainder of the polyacrylamide solution is set aside to monitor the polymerization reaction.
- After polymerization (~60 minutes) the dH2O is removed, the stacking gel soution (5% polyacrylamide) is added and the appropriate comb is inserted (check the proper comb thickness, slot number and wear safety goggles !)
- The stacking gel is allowed to polymerize
- The samples and marker are boiled for 5 min at 96ºC and collected with a quick spin
- In the meantime, the gel cassettes are assembled in the MiniCell Chambers according to the instructions of the supervisor and the inner chamber is filled with running buffer
- Upon removal of the comb, the sample slots are rinsed with running buffer using a syringe
- The samples (10 – 40 µl, depending on the slot size) are loaded via a Hamilton syringe
- Check that the inner chamber does not leak running buffer; fill up the outer compartment to the bottom of the gel holder with running buffer
- The separation is carried out at 100 V for about 1.5-2 h
- After the run is completed, disassemble the glass plates carefully and clean everything thoroughly. Use the separating part of the gel for i) Western Blotting or ii) Comassie staining