Bio-Rad PROTEAN® Plus Hinged Spacer Plates and Combs - Manual (Page 6)

2. Insert the needle into the sandwich through the gasket on the bottom of the casting stand.
3. To start the linear gradient, use the procedure described in Section 2.2, steps 6-9, except
pour the light solution into the mixing chamber and the heavy solution into the reservoir.
4. After the stirring motor is turned on, the vortex level adjusted, the valve stem and stop-
cock opened, run the peristaltic pump until the last of the acrylamide enters the needle fit-
ting. Do not allow air bubbles to enter the gel, as this could cause the mixing of the
gradient. Remove the needle from the bottom of the casting stand.
5. Overlay the gel. For a continuous gel, the comb, which should be positioned at an angel
between the gel sandwich, is straightened and inserted fully to form the wells.
6. Rinse the residual acrylamide from the gradient former, tubing, and needle as described
in Section 2.2, step. 12.
Section 3
Pouring an Exponential Gradient Gel
In some cases, the number of proteins (polypeptides) in a certain area of the gel will jus-
tify pouring an exponential gradient. Whether or not a linear gradient gives adequate resolu-
tion is determined empirically for each sample. If there are many bands near the top of the gel,
then a concave gradient is indicated, whereas if there are many bands toward the bottom of
the gel, a convex gradient should be poured. (See Theory of Linear and Exponential Gradient
Gels, Section 7, especially Figures 7.3 and 7.5). The Model 385 gradient former piston (cat-
alog number 165-2006) or the Model 395 gradient former piston (catalog number 165-2005)
limits the volume of the mixing chamber so that either a concave or a convex gradient can be
formed.
3.1 Concave Exponential Gradient Gels
Concave gradient gels are formed by delivering the acrylamide from the top, with the
small volume of heavy solution in the mixing chamber and the large volume of the light solu-
tion in the reservoir chamber. See Figure 7.2.
1. Calculate the chamber volumes.
Calculate the volume of the gel (spacer thickness in cm x gel width in cm x gel length in
cm). The volume of the heavy solution, for the mixing chamber, is one-fourth the total vol-
ume of the gel. The volume of the light solution, for the reservoir chamber, is equal to the
total volume of the gel. Because the volume of the mixing chamber is fixed, not all of
the gel solution will be delivered to the gel sandwich. One-fourth the volume will remain
in the mixing chamber at the end of the delivery and must be discarded.
2. Set up the equipment as in Section 2.2, steps 1-4.
3. Mix and degas the small volume of heavy acrylamide solution and the large volume of
light acrylamide solution, add the initiators to the light acrylamide solution, swirl 8 to 10
times, and pour into the reservoir chamber, keeping the valve stem closed.
4. Add the initiators to the heavy solution, swirl 8 to 10 times, and pour it into the mixing
chamber.
5. immediately fix the volume of the mixing chamber by inserting the piston into the cham-
ber to 1 cm above the level of the acrylamide and tightening the screw top handle to hold
the piston in place.
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