Westermeier R., Naven T., H?pker H.-R. Proteomics in Practice: A Guide to Successful Experimental Design
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Step 4: SDS Polyacrylamide Gel Electrophoresis332
.
Close the connecting valve between the reservoir
and mixing chamber.
Monomer solutions for gradient gels
.
The dense solution contains 20% glycerol to sta-
bilize the gradient.
.
The ammonium persulfate should not be added
before everything is in place, because there are
only about 10 minutes left to pour the gel until it
starts to gel.
Monomer solutions with selected % T and 3% C for 14 gradient gels
with 1 mm thickness:
Light solution, 450 mL:
8%
T
10%
T
12%
T
...... %
T
Acrylamide, Bis solution (40% T,3%C ) 90 mL 113 mL 135 mL ...... mL
1.5 mol/L Tris-Cl, pH 8.8, 0.4% SDS 113 mL 113 mL 113 mL 113 mL
TEMED (100%) 225 mL 225 mL 225 mL 225 mL
Water, deionized, make up to 450 mL 450 mL 450 mL 450 mL
Mix and degas with a water jet pump
10% ammonium persulfate 1.8 mL 1.8 mL 1.8 mL 1.8 mL
Dense solution, 450 mL:
14 %
T
16%
T
20%
T
...... %
T
Acrylamide, Bis solution (40%T,3%C) 158 mL 180 mL 225 mL ...... mL
1.5 mol/L Tris-Cl, pH 8.8, 0.4% SDS 113 mL 113mL 113 mL 113 mL
Glycerol (85%) 110 mL 110 mL 110 mL 110 mL
TEMED (100%) 225 mL 225 mL 225 mL 225 mL
Water, deionized, make up to 450 mL 450 mL 450 mL 450 mL
Mix and degas with a water jet pump
10% ammonium persulfate 1.4 mL 1.4 mL 1.4 mL 1.4 mL
For comments on amounts of TEMED and ammonium persulfate
see above on page 329.
The pinchcock clamp is closed
as well.
Add ammonium persulfate
short before gel casting.
Add ammonium persulfate
short before gel casting. Note
that less APS is added to the
dense solution!
4.1 Casting of SDS Polyacrylamide Gels 333
Control of gel polymerization for gradient gels The start of the poly-
merization is controled by the amount of ammonium persulfate. The
catalyst amount must not be too high in order to avoid overheating in
the caster. The dense solution must contain less catalyst, because the
polymerization should start at the top and proceed slowly down to the
bottom. This is very important for two reasons:
.
Thermal convection is prevented, which would
distort the gradient.
.
During polymerization the gel contracts. If the
lower portion would polymerize first, it would
pull the upper solutions down, resulting in a
curved upper edge.
Figure 4.4A shows the setup for casting gradient gels in a 14-gel cas-
ter.
.
Degas and precool the monomer solutions.
.
Add the ammonium persulfate to the monomer
solutions and mix.
.
Pour the dense solution into the reservoir.
.
Carefully open the slide valve and let just
enough solution flow to fill the connector chan-
nel. Close the valve. If too much fluid has flown
into the mixing chamber, pipette it back to the
reservoir.
.
Pour the light solution into the mixing chamber.
.
Start the magnetic stirrer.
.
Open the pinchcock clamp to the next setting,
the one next to close the tubing completely. The
light solution should start to flow down the tub-
ing.
.
Open the sliding valve of the gradient maker.
Now dense solution will flow into the mixing
chamber and will be mixed with the light solu-
tion.
Adjust the flow rate to a higher speed with the pinchcock clamp. But
the speed must not be too high to avoid mixing of solutions in the
V-shaped chamber at the bottom. Casting time should be around
10 minutes. If you estimate that it will take longer, open the pinch-
cock to the next position.
The polymerization is easier
controled with varying the
amount of APS than with
TEMED.
When conditions are correct,
the gels pull the displacing solu-
tion down.
Check for air bubbles.
Do not mix too fast; air bubbles
must be avoided.
Step 4: SDS Polyacrylamide Gel Electrophoresis334
Fig. 4.4: A. Casting gradient gels in a 14-gels caster.
B. Removing the tube and overlaying the gel solution by
spraying 0.1% SDS solution.
4.1 Casting of SDS Polyacrylamide Gels 335
.
Close the pinchcock clamp before the connec-
tion tubing runs empty, but not later than when
the liquid level reaches 3 cm below the upper
edges of the cassettes.
.
Remove the plastic tube with the connection tub-
ing from the balance chamber and rinse the
magnetic stirrer.
If monomer solution is left, fill it in a measuring cylinder; next time,
adjust the volumes of the monomers solutions to the appropriate
amount. The gels will this time have a slightly flatter gradient slope
than intended, but the separation quality will not suffer much.
The dense displacing solution will now flow down the connecting
tube and fill the V-chamber and the sloped bottom of the caster. The
gel solution level will rise to 1 cm below the cassette edges. A thin
blue layer should be visible at the bottom of the cassettes.
Should the volume of the displacing solution in the balance cham-
ber not be sufficient to produce the thin blue layer in the entire area
of the caster bottom, top more displacing solution into the balance
chamber until the blue layer has distributed over the entire bottom
area. This saves you from cutting off protruding gel pieces at the bot-
tom edge of each cassette.
.
Immediately spray 0.1% SDS solution overlay
solution over the cassettes (see Figure 4.4B).
.
Cover the caster with a cling film.
.
Let the gel polymerize overnight at room tem-
perature.
.
During unloading rinse the cassettes to remove
excess polyacrylamide.
& Note: When enough displacing solution had
been used, there is no need to cut off protruding
gel pieces at the bottom edge of each cassette.
.
Inspect each gel cassette for eventual air bub-
bles. Gels with air bubbles should not be used.
.
Store the usable gels in an airtight container
with about 100 mL gel storage solution. When
the gels are not used during the next day, place
the container with the gels into a cold room or a
refrigerator.
.
Take the foam gasket out of the front plate and
rinse it with deionized water.
.
Rinse the gel caster and the separator sheet with
mild detergent and then with deionized water.
.
Let them dry in the air.
Spraying with SDS solution is
better than pipetting water-
saturated butanol on the edge.
Because the silent polymeriza-
tion must be completed.
Do not leave it in the groove,
because it would lose its sealing
property.
Step 4: SDS Polyacrylamide Gel Electrophoresis336
4.1.4
Gel Caster for up to Six Gels
4.1.4.1 Preparation of the Gel Caster
Large gel formats require a casting box, also when only a few gels are
prepared. Otherwise the cassettes would bulge; the gels would be
thicker in the center. Never forget to place the plastic separator sheets
between the cassettes; they would firmly stick to each other after gel
polymerization. When less than 6 gels should be prepared, do not
block the non-used space with blank cassettes: cast six gels and dis-
card the non-used gels, or store them for another experiment.
Be sure that gel caster, separator sheets and cassettes are perfectly
clean and dry. The filling channel in the back wall of the caster has
originally been designed for casting homogeneous gels. However it is
much easier and faster to pour the solution directly onto the cassettes
in the caster as shown in Figure 4.5. Gradient gels are poured
through the filler port at the bottom of the front plate. For casting
homogeneous gels, leave the V-shaped rubber insert in place; for gra-
dient gels, remove it.
.
Lay the caster flat on the bench for loading.
.
Start loading with a separator sheet.
.
Place the cassettes alternating with separator
sheets into the caster, with the offset edges of
the cassettes up.
.
Assemble non-hinged cassettes outside of the
caster before you place them into the caster box.
.
Press strongly on the spacers areas on both sides
to ensure that the spacers and the glass plates
stick well together.
Finally a separator sheet and – if necessary – filling sheets are
inserted to complete the stack up to the edge of the caster. Do not
overfill it. This would cause pressure on the gels during polymeriza-
tion, when the solution starts to get warm.
.
Place the foam gasket into the groove of the
front plate. Do not cover it with grease.
.
Turn the two screws into the bottom holes and
place the front plate on the caster with the bot-
tom slots on the screws. Apply the six clamps
and tighten the screws evenly. Do not use too
much force; the sealing gasket should be com-
pressed evenly to prevent leakage.
.
For casting, place the gel caster upright in a tray,
for the occasional case that liquid is overflowing.
Dirty cassettes cause vertical
streaking in silver-stained gels.
Always take the foam gasket
out of the front plate after gel
casting.
4.1 Casting of SDS Polyacrylamide Gels 337
When the room temperature is higher than 20 C, the caster should –
like the monomer solution(s) – be pre-cooled in a cold room or a
refrigerator.
4.1.4.2 Homogeneous Gels
Very good results are usually obtained with homogeneous gels. Gradi-
ent gels need to be prepared only in special cases.
.
Close the filler port at the bottom of the front
plate with the cap or with a piece of tubing and a
pinchcock clamp.
.
For monomer solutions the ammonium persul-
fate should not be added before everything is in
place, because there are only about 10 minutes
left to pour the gel until it starts to gel.
Monomer solutions with selected % T and 3% C for six gels with
1 mm thickness, 450 mL:
7.5%
T
10%
T
12.5%
T
15%
T
Acrylamide, Bis solution (40% T,3%C ) 84 mL 113 mL 141 mL 169 mL
1.5 mol/L Tris-Cl, pH 8.8, 0.4% SDS 113 mL 113 mL 113 mL 113 mL
TEMED (100%) 225 mL 225 mL 225 mL 225 mL
Water, deionized, make up to 450 mL 450 mL 450 mL 450 mL
Mix and degas with a water jet pump
10% ammonium persulfate 1.8 mL 1.8 mL 1.8 mL 1.8 mL
For comments on amounts of TEMED and ammonium persulfate
see above on page 329.
.
Degas and precool the monomer solution.
.
Add the ammonium persulfate to the monomer
solution and mix.
.
Pour the gel solution directly into the box, avoid-
ing air bubbles (see Figure 4.5A).
.
Stop pouring when the level has reached 3 cm
below the upper edges of the casting cassettes.
.
Immediately spray 0.1% SDS solution overlay
solution over the cassettes (see Figure 4.5B).
.
Cover the caster with a cling film.
.
Let the gel polymerize overnight at room tem-
perature.
.
During unloading rinse the cassettes to remove
excess polyacrylamide.
.
Inspect each gel cassette for eventual air bub-
bles. Gels with air bubbles should not be used.
Add ammonium persulfate
shortly before gel casting.
Spraying with SDS solution is
better than pipetting water-
saturated butanol on the edge.
Because the silent polymeriza-
tion must be completed.
Step 4: SDS Polyacrylamide Gel Electrophoresis338
Fig. 4.5: A. Casting homogeneous gels in a six-gel caster.
B. Overlaying the gel solution by spraying 0.1% SDS solution.
.
Store the usable gels in an airtight container
with about 100 mL gel storage solution. When
the gels are not used during the next day, place
the container with the gels into a cold room or a
refrigerator.
.
Take the foam gasket out of the front plate and
rinse it with deionized water.
.
Rinse the gel caster and the separator sheet with
mild detergent and then with deionized water.
.
Let them dry in the air.
Monomer solution, which has been left over, is polymerized in a small
beaker before it is discarded.
4.1.4.3 Gradient Gels
For comments on gradient gels see above on page 331.
For gradient gels the monomer solution has to be introduced
through the filling port on the bottom of the front plate. In this case
we need to fill the dead volume of the V-shaped chamber in the bot-
tom of the caster with displacing solution.
.
Remove the V-shaped rubber insert from the caster.
.
Prepare 200 mL of displacing solution.
Gradient maker set up
.
Place the clean and dry gradient maker with a
magnetic stirrer on an adjustable laboratory plat-
form or on a shelf board: the level must be at
least 10 cm above the edge of the caster.
Do not leave it in the groove,
because it would lose its sealing
property.
4.1 Casting of SDS Polyacrylamide Gels 339
& Note: For reproducible gels, use always exactly
the same level for the gradient maker position.
Use always the same shelf board or measure and
note the position height of the gradient maker.
.
Place a pinchcock clamp on the flexible connec-
tion tubing, and close it completely.
.
Connect the filling port with the flexible tubing
to the gradient maker.
.
Place a magnetic stirring bar – 2 cm to 3 cm
long – into the mixing chamber.
.
Close the connecting valve between the reservoir
and mixing chamber.
Monomer solutions for gradient gels
.
The dense solution contains 20% glycerol to sta-
bilize the gradient.
.
The ammonium persulfate should not be added
before everything is in place, because there are
only about 10 minutes left to pour the gel until it
starts to gel.
Monomer solutions with selected % T and 3% C for 6 gradient gels
with 1 mm thickness:
Light solution, 211 mL:
8%
T
10%
T
12%
T
... %
T
Acrylamide, Bis solution (40% T,3%C ) 42mL 53mL 63mL ...mL
1.5 mol/L Tris-Cl, pH 8.8, 0.4% SDS 53 mL 53 mL 53 mL 53 mL
TEMED (100%) 105 mL 105mL 105 mL 105 mL
Water, deionized, make up to 210 mL 210 mL 210 mL 210 mL
Mix and degas with a water jet pump
10% ammonium persulfate 840 mL 840 mL 840 mL 840 mL
Dense solution, 211 mL:
14%
T
16%
T
20%
T
... %
T
Acrylamide, Bis solution (40% T,3%C ) 74 mL 84 mL 105 mL ... mL
1.5 mol/L Tris-Cl, pH 8.8, 0.4% SDS 53 mL 53 mL 53 mL 53 mL
Glycerol (85%) 50 mL 50 mL 50 mL 50 mL
TEMED (100%) 105 mL 105 mL 105 mL 105 mL
Water, deionized, make up to 210 mL 210 mL 210 mL 210 mL
Mix and degas with a water jet pump
10% ammonium persulfate 670 mL 670 mL 670 mL 670 mL
The pinchcock clamp is closed
as well.
Add ammonium persulfate
shortly before gel casting.
Add ammonium persulfate
short before gel casting. Note
that less APS is added to the
dense solution!
Step 4: SDS Polyacrylamide Gel Electrophoresis340
For comments on amounts of TEMED and ammonium persulfate
see above on page 329.
Control of gel polymerization for gradient gels The start of the poly-
merization is controled with the amount of ammonium persulfate.
The catalyst amount must not be too high in order to avoid overheat-
ing in the caster. The dense solution must contain less catalyst,
because the polymerization should start at the top and proceed slowly
down to the bottom. This is very important for two reasons:
.
Thermal convection is prevented, which would
distort the gradient.
.
During polymerization the gel contracts. If the
lower portion would polymerize first, it would
pull the upper solutions down, resulting in a
curved upper edge.
Figure 4.6 shows the setup for casting a gradient gel in the caster for
six gels.
.
Degas and precool the monomer solutions.
.
Add the ammonium persulfate to the monomer
solutions and mix.
.
Pour the dense solution into the reservoir.
.
Carefully open the slide valve and let just
enough solution flow to fill the connector chan-
nel. Close the valve. If too much fluid has flown
into the mixing chamber, pipette it back to the
reservoir.
.
Pour the light solution into the mixing chamber.
.
Start the magnetic stirrer.
.
Open the pinchcock clamp to the next setting,
the one next to close the tubing completely. The
light solution should start to flow down the tub-
ing.
.
Open the sliding valve of the gradient maker.
Now dense solution will flow into the mixing
chamber and will be mixed with the light solu-
tion.
Adjust the flow rate to a higher speed with the pinchcock clamp. But
the speed must not be too high to avoid mixing of solutions in the V-
shaped chamber at the bottom. Casting time should be around 10
minutes. If you estimate that it will take longer, open the pinchcock
to the next position.
The polymerization is more
easily controled with varying
the amount of APS than with
TEMED.
When conditions are correct,
the gels pull the displacing solu-
tion down.
Check for air bubbles.
Do not mix too fast; air bubbles
must be avoided.
4.1 Casting of SDS Polyacrylamide Gels 341
Fig. 4.6: A. Casting gradient gels in a 6-gels caster.
B. Overlaying the gel solution by spraying 0.1% SDS solution.
.
Close the pinchcock clamp before the connec-
tion tubing runs empty.
.
Close the sliding valve between the mixing
chamber and the reservoir.
.
Pour 200 mL displacing solution into the mixing
chamber of the gradient maker.
.
Open the pinchcock clamp to the same position
like casting the gradient.
.
When the monomer solutions have reached the
desired level, which is 1 cm below the upper
edges of the cassettes, close the pinchcock
clamp.
The displacing solution – visible because of the added Bromophenol
blue – should fill the V-shaped chamber completely. This saves you
from cutting off protruding gel pieces at the bottom edge of each cas-
sette.
.
Immediately spray 0.1% SDS solution overlay
solution over the cassettes (see Figure 4.6B).
.
Cover the caster with a cling film.
.
Let the gel polymerize overnight at room tem-
perature.
.
During unloading rinse the cassettes to remove
excess polyacrylamide.
.
Inspect each gel cassette for eventual air bub-
bles. Gels with air bubbles should not be used.
Spraying with SDS solution is
better than pipetting water-
saturated butanol on the edge.
Because the silent polymeriza-
tion must be completed.