Exercise 4.1 - Preparation of SDS-Polyacrylamide Gels
Figure 4.5 Formed slab gel in casting unit
- 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.
- 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
- Turn off the vacuum, open the flask and add 200 µl of ammonium
persulfate and 20 µl 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 two
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
- 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 µl ammonium persulfate and 10
µl 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 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
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.
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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org