SDS-PAGE Protocol

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SDS-PAGE Protocol


  1. Prepare 1L of 1x running buffer from the 10x running buffer solution.
  2. 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.
  3. Allow to polymerize for 50 min. Sunlight helps.
  4. 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.
  5. When the gel is polymerized, the comb may be removed gently. Load the gel (keep between plates), into the electrophoresis apparatus.
  6. Boil samples in sample buffer for 10 min to denature. Spin down at 10000 rmp for 10 sec to pool sample.
  7. Load the molecular weight ladder into the first lane of the gel- 5uL.
  8. Load your sample into the gel- 10-20uL.
  9. Fill the inner reservoir with 1x running buffer and fill the outside to the appropriate fill line.
  10. Place the top on the container.
  11. Apply the power source- 160-200V. Stop when the dye just reaches the bottom.
  12. Under water, peel plates apart and remove gel. The running water is used to prevent tearing.
  13. Rinse gel with water 2x.
  14. Add Coomassie Brilliant Blue stain to the gel (enough to completely cover gel).
  15. Let stain for ~30 minutes.
  16. Remove Commassie Stain and rinse with DI water.
  17. Let soak overnight in destain/water. Add 3 balled up kimwipes to absorb dye and speed up the destaining process.
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