Team:TU Darmstadt/protocols/SDS-Page

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Labbook | Matierals | Protocols

Protocols


Equipment

  • - Two glass plates
  • - Gel caster
  • - Comb
  • - VWR Power Source 300V
  • - Heat block


Chemicals & consumables

  • - SDS
  • - Rotiphorese® (30%)
  • - Tris HCl
  • - Glycine
  • - TEMED
  • - APS
  • - Aqua dest.
  • - Isopropyle alcohol
  • - Glycerine
  • - Beta-Mercaptoethanol
  • - Bromphenolblue
  • - Coomassie brilliant blue G250
  • - Coomassie brilliant blue R250
  • - Methanol
  • - Acetate (99%)


Buffers & gels

  • - Separation buffer (0.5 M Tris, 0.4% SDS, pH = 8.8)
  • - Stacking buffer (0.5 M Tris, 0.4% SDS, pH = 6.6)
  • - Running buffer (0.25 M Tris, 2 M glycine, 1% SDS , pH = 8.3)
  • - Separation gel 12.5% (5 ml Separation buffer, 6.25 ml Rotiphorese®, 3.75 ml Aqua dest., 30 µl TEMED, 30 µl APS (40%))
  • - Stacking gel 4% (3 ml Separation buffer, 1.33 ml Rotiphorese®, 5.67 ml Aqua dest.,20 µl TEMED, 20 µl APS (40%)
  • - 3x Sample buffer (65 mM Tris, 4% SDS, 20% glycerine, 10% beta-Mercaptoethanol, 1 tip of bromphenolblue, pH = 6.75)
  • - Staining buffer (0.5 g Coomassie brilliant blue G250 & R250 each, 100 ml methanol, 100 ml Aqua dest., 20 ml acetate)
  • - Destaining buffer (400 ml Aqua dest., 100 ml methanol, 30 ml acetate)


Procedure


  1. Load and Run
    2.
  2. prepare the separating gel and fill it into the chamber
  3. pour 1 mL isopropyle alcohol on the top of the gel to destroy air bubbles and prevent dehydration
  4. discard the isoprpyl alcohol and pour the prepared stacking gel
  5. stick in the comb
  6. if not used, store the gel in wet cloth (to prevent dehydration) at 4°C
  7. if used, remove comb when the gel is solid and place it into SDS PAGE chamber
  8. fill chamber with running buffer
  9. after heating the samples with 3x sample buffer at 70°C for 15 min, apply 20 µl to each pocket
  10. load most outer pocket with a commercial protein marker
  11. start the PAGE by applying 20 mA / gel at stacking
  12. apply 40 mA / gel at separation

  13. Staining of gel
    14.
  14. prepare the separating gel and fill it into the chamber
  15. pour 1 mL isopropyle alcohol on the top of the gel to destroy air bubbles and prevent dehydration
  16. discard the isoprpyl alcohol and pour the prepared stacking gel
  17. stick in the comb
  18. if not used, store the gel in wet cloth (to prevent dehydration) at 4°C
  19. if used, remove comb when the gel is solid and place it into SDS PAGE chamber
  20. fill chamber with running buffer
  21. after heating the samples with 3x sample buffer at 70°C for 15 min, apply 20 µl to each pocket
  22. load most outer pocket with a commercial protein marker
  23. start the PAGE by applying 20 mA / gel at stacking
  24. apply 40 mA / gel at separation

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