Berglund:SDS-PAGE

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Contents

SDS-PAGE Gels

The old, longer protocol can be found here.


Separating Gel

Separating Gel
10%13%15%18%
2 gels3 gels4 gels3 gels4 gels2 gels4 gels2 gels4 gels
40% (29:1) Acrylamide 2mL3mL4mL4mL5.3mL3mL6mL3.6mL7.2mL
1M Tris pH 8.9 3.12mL4.68mL6.24mL4.68mL6.24mL3.12mL6.24mL3.12mL6.24mL
ddH2O 2.8mL4.2mL5.6mL3.2mL4.3mL1.8mL3.6mL1.2mL2.4mL
10% SDS 83.3μL125μL166.6μL120μL166.6μL83.3μL166.6μL83.3μL166.6μL
Total volume 8mL12mL16mL12mL16mL8mL16mL8mL16mL
  1. Mix separating gel.
  2. Add 50μL fresh ammonium persulfate and 16μL TEMED. (More TEMED = faster polymerization).
  3. Pour 4mL per gel (up to the bottom of the green bar) using P1000 pipette.
  4. Layer 2-methyl-1-propanol (isobutanol) or isopropanol on top.
  5. Leave at room temperature for >30 minutes to polymerize.
  6. Wash out isobutanol with distilled H2O, dry with strip of Whatman paper.

Stacking Gel

Stacking Gel
2 gels3 gels4 gels
40% (29:1) Acrylamide 0.5mL0.735mL0.98mL
1M Tris pH 6.8 0.6mL0.875mL1.16mL
ddH2O 3.5mL5.24mL6.9mL
10% SDS 50μL75μL100μL
Total volume 4.6mL7mL9.3mL
  1. Mix stacking gel.
  2. Add 50μL fresh ammonium persulfate and 12μL TEMED.
  3. Pour ~2mL per gel (to nearly the top of the plates) using P1000 pipette.
  4. Insert combs, clean spills.
  5. Leave at room temperature for >30 minutes to polymerize.

To use immediately

  • Put into gel box with small plate facing core of the box.
  • Lock in the plates so there are no leaks.
  • Pour 1x SDS running buffer into the core of the box so that it spills over the glass plates and into the base of the box.
  • Carefully remove combs by pulling straight up.
  • Use a pipette tip to straighten any crooked wells.

To store for later use

  • Remove plates from rack.
  • Remove comb
  • Wrap tightly with saran wrap with lightly moistened paper towel in between plates.
  • Label with name, date, and gel percentage and store at 4°C.

PLEASE don't store plates with combs, we don't have enough combs for long term storage!

Buffers

Loading buffer/dye

  1. Mix sample with dye, 1:4 for 5x, 1:1 for 2x. Boil at 100°C for 2 min.
  2. Run gel at 8milliamps until dye has reached the separating gel then at 25milliamps until dye has reached the bottom of the gel.

10X SDS Running buffer*

  • 1134g Glycine
  • 240g Tris base
  • Add to 6L of DDH2O while stirring.
  • After dissolved, bring final volume to 8L.
  • pH to 8.8, store at room temperature.

1XSDS Running Buffer*

  • Make fresh from 10X (1L of 10x into 9L of water)
  • Add 20% SDS to give final concentration of 0.1% (50ml into 10L)

*These are no longer the buffer protocols we use. Will be updated someday.

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