Berglund:SDS-PAGE

SDS-PAGE Gels

The old, longer protocol can be found here.

Separating Gel

Separating Gel

	10%			13%		15%		18%
	2 gels	3 gels	4 gels	3 gels	4 gels	2 gels	4 gels	2 gels	4 gels
40% (29:1) Acrylamide	2mL	3mL	4mL	4mL	5.3mL	3mL	6mL	3.6mL	7.2mL
1M Tris pH 8.9	3.12mL	4.68mL	6.24mL	4.68mL	6.24mL	3.12mL	6.24mL	3.12mL	6.24mL
ddH2O	2.8mL	4.2mL	5.6mL	3.2mL	4.3mL	1.8mL	3.6mL	1.2mL	2.4mL
10% SDS	83.3μL	125μL	166.6μL	120μL	166.6μL	83.3μL	166.6μL	83.3μL	166.6μL
Total volume	8mL	12mL	16mL	12mL	16mL	8mL	16mL	8mL	16mL

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 gels	3 gels	4 gels
40% (29:1) Acrylamide	0.5mL	0.735mL	0.98mL
1M Tris pH 6.8	0.6mL	0.875mL	1.16mL
ddH2O	3.5mL	5.24mL	6.9mL
10% SDS	50μL	75μL	100μL
Total volume	4.6mL	7mL	9.3mL

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.