Lidstrom: SDS-PAGE

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Gel Prep

• Clean cover plate and thicker spacer plate 75 mM
  • Soap and Water
  • Ethanol
  • DI water
• Dry plates
• Setup one spacer plate and one cover plate in each gel holder
  • The cover plate goes on the side of the spacer plate with the spacers in order to create a small gap between the plates
• Put the gel holder into the casting stand

Pour the Resolving Gel

• Mix components of the amounts in the Gel Mix link for the resolving gel (Recipes is for 4 gels). Mix in the order listed.
• Gel Mix Recipe
  • Don't add APS/TEMED until ready to pour
• Use pipette to put gel mix into the gap between the plates
• Carefully layer 50%EtOH 50% ddH2O on top of the gel to prevent the top of the gel from drying out
• Let dry for an hour
• Store at 4 deg C wrapped in a wet paper towel and saran wrap if you're not going to use it right away.

Pour the Stacking Gel

• Replace gel in gel holder
• Dry surface of gel carefully with Kimwipe or paper towel
  • It can be a little gooey
• Mix components of the amounts in the Gel Mix link. Mix in the order listed.
• Gel Mix Recipe
  • Don't add APS/TEMED until ready to pour
• Use pipette to put gel mix into the gap between the plates
• Insert the comb being 'careful not to trap any bubbles'
• Attach binder clips to help hold the comb in while drying. One in either side of the casting stand clamp.

binder clips squeeze the glass to the comb. Put them as far down as they go.

• Let dry for an hour

Sample Prep

• 10-20 ug protein
• Load 12-15 uL, absolute max 20 uL for big comb
• Denature protein
  • Mix with sample buffer & BME
  • Boil for 5-10 min (can do longer times at lower temp, too.)

Running the Gel

• Bio-Rad Mini-Cell Setup
  • If only 1 gel, use buffer dam to replace second gel
• Slot gels with cover plates facing each other...
• Apply pressure on gel holder and gels as you close the tabs to seal the center compartment.
• Fill central compartment with running buffer
• Pour rest into outer compartment
• Load gel
• Make sure to color/charge-match the cords to the power unit as the electrodes in the gel holder to the contacts in the lid.
• Run 30-45 min @250V

Other Resources

• Background
• Instructions for PageRuler protein standard