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
  • The APS solution should be ~ 1-2 months old. A 3 month old solution failed to cause polymerization -JM 10/2012
• 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.
• Leave for 1 hour while polymerization occurs.
• Can store for a few weeks in the fridge. Leave comb in, and wrap in a wet paper towel and cling wrap.

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

Sample Prep

• 10-20 ug protein
• Load 12-15 uL, absolute max 20 uL for big comb
• Denature protein
  • Mix with sample buffer & BME
    • How much?
  • 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.

Mini-cell Gel holder

• 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
  • Amanda runs 20 min at 200V, then checks frequently to make sure you don't run your protein off the gel.

Staining, Destaining, & Visualization

• dye overnight or cycles of 1 min @ power 6 in the microwave
  • microwave by Bo's bench. Let it vent a little in the hood between heating events.
• Return dye to container
• Rinse to remove residual dye
• Destain (I do 2 rounds at least)

Other Resources

• Background (Wikipedia)
• Instructions for PageRuler protein standard
• Instructions for BioRad Rainbow Std

Mistakes to Be Careful About

• letting the gel dry too long after pouring the stacking gel (comb step)
  • the very edges can shrivel up, which becomes a problem when you try to use those edge lanes
• sample sloshing out of the well you are using into a neighboring well

Recipes

• Loading Buffer (5x):
  • 10% w/v SDS, 10 mM Dithiothreitol or beta-mercapto-ethanol, 20 % v/v Glycerol, 0.2 M Tris-HCl, pH 6.8, 0.05% w/v Bromophenolblue
  • Should add up to 8M urea for really hydrophobic proteins
  • To make this buffer, add:
    • blah

• Running Buffer:
  • 10x SDS running buffer (1 liter): 30.2 g Tris-HCl, 144 g Glycine, 10 g SDS, adjust to pH to 8.3; bring volume up to 1L with dH20; store at room temperature. Dilute to 1x with dH20.
  • This buffer is used while running proteins through the gel. Pour it in as the instructions for the box explain. Pour back into bottle for re-use afterward.
  • Keep until ____; remake after this.

• Staining Buffer(Coomassie Brilliant Blue G-250):
  • 0.500 g Brilliant Blue, 500 mL methanol, 100 mL glacial acetic acid, 400 mL dH20. Mix well. Store at room temperature; can be reused 2-3 times.
  • note: Coomassie Brilliant Blue G-250 differs from Coomassie Brilliant Blue R-250 by the addition of two methyl groups. We use the G form. Read more about the R form here.

• Destaining Buffer:

Acrylamide toxicity

• Acrylamide is toxic to your nervous system, and may be a carcinogen. The unpolymerized form is toxic, but the polymerized form is much less toxic. ALWAYS wear gloves and wipe up spills - once the solution drys, the dust can be inhaled. Interestingly, fried starchy/sugary foods naturally contain acrylamide, too.
• more than you want to know about acrylamide toxicity can be found here