SDS-PAGE

Pouring SDS-Polyacrylamide gels

Reagents

• 30% acrylamide mix *
• 1.5M Tris (pH 8.8)
• 1.0M Tris (pH 6.8)
• 10% SDS
• 10% Ammonium persulfate (made up directly before use)
• TEMED

*unpolymerized acrylamide is a neurotoxin – use gloves

1. Assemble the glass plates in the holder with the plastic sheets in between each set of glass plates: start with the open casting chamber body face up on the benchtop, the thumbscrews should be facing the ceiling.
2. First add a separation sheet so that it sits at the bottom, then add a spacer plate (spacer side up) on top of the separation sheet. Then add a short plate on top. Make sure that each addition is seated at the bottom of the chamber. Repeat, until all 9 plate sets have been assembled. Add 3 acrylic blocks and then sufficient separation sheets until the stack is flush with the edge of the chamber.
3. To ensure a good seal the entire stack should be flush as possible to the top of the casting chamber, and not extend beyond it. If you overfill the chamber, monomer solution may leak out during pouring. Seat the gasket firmly in the notch in the sealing plate. With the screws loosened, slide the sealing plate under the clamps of the casting chamber, being careful not to disturb the stack. The inlet ort should match the groove at the bottom of the chamber. Gradually tighten the screws in a random fashion until tight. Test seal with water – open valve to remove water, close again before casting gels. Stand chamber up and place on a level surface. Do not invert chamber at this stage.
4. Determine the % of the resolving gel to be poured, use Table 1 as a guide for how much of each reagent to add.
5. Prepare two Erlenmeyer flasks, one for the resolving gel and one for the stacking gel.
6. Add all components to each flask except for the TEMED and the APS (polymerization will begin very soon after the addition of TEMED and APS
7. Add the TEMED and APS to the resolving gel flask, swirl all components and then rapidly proceed to the next step
8. Using a Pasteur pipette, or a 10ml pipette, add the resolving gel solution in between the glass plates. Leave sufficient space (the length of the comb plus ~1cm for the stacking gel)
9. Using a Pasteur pipette, carefully overlay the solution with isobutanol (or water)
10. Use a Pasteur pipette to aspirate the remaining solution from the Erlenmeyer flask. Once this has solidified (20-30 minutes), move on to the next step.
11. Pour off the overlay and blot on some paper
12. Finish preparing the stacking gel by adding the TEMED and APS.
13. Swirl the mixture and add to the top of the resolving gel using a Pasteur pipette. Fill to approximately 1cm below the top.
14. Rapidly add the comb into each set of glass plates
15. Allow the gel to set
16. Once gels have set, unscrew the clamps and remove the sealing plates and acrylic blocks. Remove gels and rinse with distilled water. Trim off excess acrylamide with a razor blade.
17. Store the gels at 4 C in TGS buffer (Tris-glycine with SDS see below)

• 25mM Tris
• 250mM glycine (pH 8.3)
• 0.1% SDS

A 10X stock can be made by dissolving 30.2g of Tris base and 188g of glycine in 900ml of milliQ water. Then 50ml of a 20% SDS solution can be added and the volume adjusted to 1000ml with water.

• 2.4ml of1M Tris-HCl, pH 6.8
• 3ml of 20% SDS
• 3ml of glycerol
• 1.6ml of beta-mercaptoethanol
• 6mg of bromophenol blue

• 45% Methanol (reagent grade)
• 10% Glacial acetic acid
• 45% Water
• 3g/L Coomassie Brilliant Blue R250

1) Add 100mL of glacial acetic acid to 450mL ultrapure water.

2) Dissolve the 3g of Coomassie Dye in 450mL methanol.

3) Filter the solution before use

• 50% Methanol (reagent grade)
• 10% Glacial acetic acid
• 40% Water