Exercise 4.1 - Preparation of SDS-Polyacrylamide Gels

LEVEL I

Figure 4.5 Formed slab gel in casting unit

Materials

• Casting gel unit for electrophoresis
• Siliconized pasteur pipettes
• Syringes equipped with blunt, stub nosed, needles 6
• Vacuum chamber for degassing gels.
• Micropipettes (10-300 µl)
• Stock 30%T:0.8%C Acrylamide monomer
• 1.5 M Tris-HCl buffer, pH 8.8
• 10% (w/v) SDS
• 10% (w/v) Ammonium persulfate

  Temed acrylamide is a powerful neurotoxin. Do not breathe powder or otherwise come in contact with the monomer. Wear gloves at all times.

• Separation Gel Mixed Just Prior To Use
  • 20 ml of Acrylamide monomer
  • 15 ml of Tris-HCl Buffer, pH 8.8
  • 0.6 ml of 10% (w/v) SDS
  • 24.1 ml of HO
• Stacking gel mixed just prior to use
  • 2.66 ml of Acrylamide monomer
  • 5.0 ml of Tris buffer, pH 8.8
  • 0.2 ml of 10% (w/v) SDS
  • 12.2 ml of HO

Procedure

1. Assemble your slab gel unit with the glass sandwich set in the casting mode, and with 1.5 mm spacers in place. Refer to Figure 4.5.

2. Prepare a separating gel from the ingredients listed above.

3. Add the separating gel to a side arm flask, stopper the flask and attach to a vacuum pump equipped with a cold trap. Turn on the vacuum and degas the solution for approximately 10 minutes. During this period, gently swirl the solution in the flask. 7

4. Turn off the vacuum, open the flask and add 200 µl of ammonium persulfate and 20 µl of TEMED to the solution.

5. Add the stopper to the flask and degas for an additional 2 minutes while gently swirling the solution to mix the two accelerators. Use this solution within a few minutes of mixing, or it will gel in the flask.

6. Transfer the degassed acrylamide solution to the casting chamber with a pasteur pipette. Gently fill the center of the glass chamber with the solution by allowing the solution to run down the side of one of the spacers. Be careful not to introduce air bubbles during this step.

7. Adjust the level of the gel in the chamber by inserting a syringe equipped with a 22 gauge needle into the chamber and removing excess gel.

8. Immediately water layer the gels to prevent formation of a curved meniscus.
   Using a second syringe and needle, add approximately 0.5 ml of water to the chamber by placing the tip of the needle at an angle to a spacer and gently allowing the water to flow down the edge of the spacer and over the gel. Add an additional 0.5 ml of water to the chamber by layering it against the spacer on the opposite side of the chamber. Done appropriately, the water will form a layer over the gel, and a clear line of demarcation will be observed as the gel polymerizes. 8

9. After 30 minutes, the gel should be polymerized. If degassing was insufficient, or the ammonium persulfate not fresh, the polymerization may take an hour or more. When the gel is polymerized, lift the gel in its casting chamber and tilt to decant the water layer.

10. Prepare a stacking gel from the listed ingredients.

11. Degas the stacking gel as in step 3.

12. Add 75 µl ammonium persulfate and 10 µl TEMED to the stacking gel and degas for an additional 2 minutes.

13. Add approximately 1 ml of stacking gel to the gel chamber and gently rock back and forth to wash the surface of the separating gel. Pour off the still liquid stacking gel and dispose of properly. Remember that liquid acrylamide is extremely hazardous!

14. Add fresh stacking gel to nearly fill the chamber, but allow room for the insertion of a teflon comb used to form sample wells. Carefully insert a teflon comb into the chamber. 9 Adjust the volume of the stacking gel as needed to completely fill the spaces in the comb.
    Be careful not to trap any air bubbles beneath the combs. Oxygen inhibits polymerization, and will subsequently result in poor protein separations.

15. Allow the gels to polymerize for at least 30 minutes prior to use.

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