- Casting gel unit for electrophoresis
- Siliconized Pasteur pipettes
- Syringes equipped with blunt stub-nosed needles
- Vacuum chamber for degassing gels
- Micropipettes (10–300 mL)
- 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 H2O.
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 H2O
- Assemble your slab gel unit with the glass sandwich set in the casting
mode with 1.5-mm spacers in place.
- Prepare a separating gel from the ingredients listed.
- 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.
- Turn off the vacuum, open the flask, and add 200 mL of ammonium
persulfate and 20 mL of TEMED to the solution.
- Add the stopper to the flask and degas for an additional 2 minutes while
gently swirling the solution to mix the 2 accelerators. Use this solution
within a few minutes of mixing, or it will gel in the flask.
- 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.
- 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.
- 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.
- 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.
- Prepare a stacking gel from the listed ingredients.
- Degas the stacking gel as in step 3.
- Add 75 mL of ammonium persulfate and 10 mL of TEMED to the stacking
gel and degas for an additional 2 minutes.
- 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!
- Add fresh stacking gel until it nearly fills 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. 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.
- Allow the gels to polymerize for at least 30 minutes prior to use.