Bio-Rad PROTEAN Plus Multi-Casting Chamber - Manual (Page 9)

Note: For the larger format gels (
≥ 20cm in width) it is necessary to decrease the TEMED
concentration by 50% (for a final concentration of 0.025%). This will extend the polymerization
time to approximately 20 minutes, providing time to complete casting and for the monomer
solution to settle properly. Also, adding 10% GLYCEROL to the HEAVY solution is important
when preparing GRADIENT GELS. Glycerol adds "weight" to the heavy solution, the solution
that flows into the multi-casting chamber behind the light solution. This added weight prevents
the heavy solution from pushing up through the middle into the light solution. (This would cause
a ripple in the gradient.) Glycerol is washed out during regular staining and protein digestion
procedures which use acetonitrile and ammonium bicarbonate. It will not interfere with
protein identification (using mass spectrometry).
Sample Calculation: Linear Gradient Gels
Objective: Cast 12 gels, 2.0 mm, 25 x 20.5 cm, 8–16% acrylamide.
Place 12 clean hinged spacer plates and separation sheets in the multi-casting chamber
(as described in Section 2) and measure the volume required to fill the plates with water.
In this example, the volume required is 1330 ml. Disassemble and dry all components.
Prepare 1340 ml of solution, 675 ml of light monomer solution (8%) and 665 ml of heavy
monomer solution (16.5%). Use the standard equation, C
1
V
1
= C
2
V
2
.
To prepare 675 ml (8%)
(8%) (675 ml) = (X ml) (30% acrylamide/Bis Stock) 180 ml
(0.375M Tris-Cl)(675 ml) = (X ml) (1.5M Tris-Cl pH 8.8 Stock) 169 ml
(675 ml) – (180 ml + 169 ml) = ml Water 326 ml
(0.05% APS) (675 ml) = (X ml) (10% APS Stock) 3.375 ml
(0.025% TEMED) (675 ml) = (X ml) (100% TEMED Stock) 169 µl
To prepare 665 ml (16.5%)
(16.5%) (665 ml) = (X ml) (30% acrylamide/Bis Stock) 366 ml
(0.375M Tris-Cl)(665 ml) = (X ml) (1.5M Tris-Cl pH 8.8 Stock) 166 ml
(10% GLYCEROL) (665 ml) = (X ml) (100% GLYCEROL) 66.5 ml
(665 ml) – (366 ml + 166 ml + 66.5 ml) = ml Water 66.5 ml
(0.05% APS) (665 ml) = (X ml) (10% APS Stock) 3.325 ml
(0.025% TEMED) (665 ml) = (X ml) (100% TEMED Stock) 166 µl
3.2.3. Combine all reagents except the initiators (usually APS and TEMED) and degas the
solutions for 15 minutes under a vacuum. Degassing the solutions removes oxygen
(oxygen inhibits polymerization.)
3.2.4. Immediately prior to pouring, add TEMED and APS to both solutions, mix gently,
and pour the appropriate monomer solutions into the gradient chambers. The light
solution (the one with the lower acrylamide concentration) should be placed in the
mixing chamber labeled "light", and the heavy solution in the reservoir chamber labeled
"heavy". The level of the solutions in the two chambers will NOT be equal at this time.
3.2.5. Turn on the stirring bar in the mixing chamber to a steady speed and maintain this
same speed throughout casting.
Note: If flow rate is kept constant, gradients will be reproducible. Tubing size, the volume of
acrylamide in the chambers, and the rate of stirring must be kept constant. If the rate of
stirring changes, the vortex that is created will also vary. The speed controls the amount of
acrylamide pulled into the mixing (light) chamber from the reservoir (heavy) chamber when
the connection between the two chambers is opened. Variation in stirring can disrupt the
linear gradient formation. Also, the vortex should be off-center inside the mixing chamber.
This can be achieved by moving the gradient former on the stir plate to offset the stir bar.
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