Bio-Rad PROTEAN Plus Multi-Casting Chamber - Manual (Page 10)

3.2.6. Start casting the gels by opening the STOPCOCK valve to the multi-casting chamber
and the STOPCOCK valve to the gradient former. The valve stem lever on the
gradient former is closed at this time. Allow the light monomer solution to enter the
multi-casting chamber until the level of solution in the mixing chamber is EQUAL to
the level of the heavy monomer solution in the reservoir chamber. The light solution
should be close to entering the plates in the casting chamber. This should help
compensate for the V-groove in the multi-casting chamber.
Note: The gradient must be poured as quickly as possible, without mixing the gradient
solution in the casting chamber. In addition to decreasing the amount of initiators, a peristaltic
pump can be used to pump the entire set of gradients within 10 minutes. However, if it is still
not possible to complete the operation in 10 minutes with a pump, decrease the concentration
of initiators to slow polymerization. An inexperienced user should practice all steps ahead of
time so that the procedure of starting the gradient is completed quickly.
Note: The larger format gels (
≥ 20cm in width) require a decrease in the TEMED concentration
by 50% (for a final concentration of 0.025%).
3.2.7. Open the VALVE STEM lever on the gradient former once the levels are EQUAL in
the two chambers, and begin mixing the solutions and creating the gradient. DO NOT
allow any air bubbles to enter the plates.
3.2.8. After casting the gels to the top of the plates, CLOSE the stopcock on the multi-
casting chamber and on the gradient former. Carefully remove the luer connector from
the stopcock connected to the multi-casting chamber. Put the tubing into a beaker and
drain the excess acrylamide from the gradient former. Insert the combs at an angle;
this will help minimize bubble formation in the wells.
Caution: Immediately flush the gradient former and tubing with water to prevent
polymerization of residual acrylamide within the gradient former.
Section 4
Removing Gels, Storage and Use of the Gels
Note: Wear rubber gloves while performing the following procedure to prevent accidental
exposure to unpolymerized acrylamide, a neurotoxin.
1. Allow at least one hour for polymerization. After polymerization, remove the sealing
plate from the multi-casting chamber. Remove the gels from the stack one at a time using
the green gel releaser. Place the gel releaser between two plates on the hinged side. Pull
the gel releaser forward, pushing against the back plate still inside the chamber. This
should push and move one plate forward and release it from the rest of the stack. Lift the
hinged spacer plate out of the chamber. Trim off excess acrylamide around the glass
edges with the gel releaser.
Note: Polymerized acrylamide will be between the plates, and adhered to the plates,
separation sheets and edges of the chamber. Use the gel releaser to remove excess acrylamide.
2. Rinse off the tops of all the gels thoroughly with distilled water. Wash off any pieces of
excess acrylamide present with distilled water.
3. Store the gels upright in a tightly sealed container or zip-lock bag. Add a few milliliters
of 1 X gel buffer (identical to the buffer in the gel) to the bottom of the container and to
the tops of the gels to prevent them from drying out. Store tightly sealed at 4 °C.
Note: If a stacking gel is required, it should be cast immediately prior to use.
4. Clean the entire casting chamber thoroughly with distilled water. Residual acrylamide in
the stopcock valve and inlet port can be removed using a pipette tip.
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