- Prepare 1L of 1x running buffer from the 10x running buffer solution.
- Create resolving gel mixture. Add APS last. Once APS is added, fill the gel plates up to 1cm below the max depth of the comb.
- Allow to polymerize for 50 min. Sunlight helps.
- Create stacking gel mixture. Add APS to the solution according to the SDS-PAGE solution guide and immediately pour into the gel set up. Fill to near the top but leave a small space to place the comb in the gel. Then insert the comb and allow the gel to set- 20 min.
- When the gel is polymerized, the comb may be removed gently. Load the gel (keep between plates), into the electrophoresis apparatus.
- Boil samples in sample buffer for 10 min to denature. Spin down at 10000 rmp for 10 sec to pool sample.
- Load the molecular weight ladder into the first lane of the gel- 5uL.
- Load your sample into the gel- 10-20uL.
- Fill the inner reservoir with 1x running buffer and fill the outside to the appropriate fill line.
- Place the top on the container.
- Apply the power source- 160-200V. Stop when the dye just reaches the bottom.
- Under water, peel plates apart and remove gel. The running water is used to prevent tearing.
- Rinse gel with water 2x.
- Add Coomassie Brilliant Blue stain to the gel (enough to completely cover gel).
- Let stain for ~30 minutes.
- Remove Commassie Stain and rinse with DI water.
- Let soak overnight in destain/water. Add 3 balled up kimwipes to absorb dye and speed up the destaining process.
- Optional: add Coomassie Destain Solution to the gel and kimwipes abs let sit for several hours, then replace Destain with water