Protein Protocols Protein Protocols

Polyacrylamide Gel Electrophoresis of Proteins 71
9. Degas this mixture under vacuum for 30 s and then add TEMED (12 µL).
10. Pour off the water overlayering the gel and wash the gel surface with about 2 mL of
stacking gel solution and then discard this solution.
11. The gel slab is now filled to the top of the plates with stacking gel solution and the wellforming
comb placed in position (see Chapter 11).
12. When the stacking gel has set (~15 min), carefully remove the comb. The gel is now ready
for running. The conditions of running and sample preparation are exactly as described for
SDS gel electrophoresis in Chapter 11.
4. Notes
Fig. 1. Gradient forming apparatus.
1. The total volume of liquid in reservoirs A and B should be chosen such that it approximates
to the volume available between the gel plates. However, allowance must be made
for some liquid remaining in the reservoirs and tubing.
2. As well as a gradient in acrylamide concentration, a density gradient of sucrose (glycerol
could also be used) is included to minimize mixing by convectional disturbances caused
by heat evolved during polymerization. Some workers avoid this problem by also including
a gradient of ammonium persulfate to ensure that polymerization occurs first at the top
of the gel, progressing to the bottom. However, we have not found this to be necessary in
our laboratory.
3. The production of a linear gradient has been described in this chapter. However, the
same gradient mixed can be used to produce a concave (exponential) gradient. This concave
gradient provides a very shallow gradient in the top half of the gel such that the
percentage of acrylamide only varies from about 5–7% over the first half of the gel.
The gradient then increases much more rapidly from 7–20% over the next half of the gel.
The shallow part of the gradient allows high-mol-wt proteins of similar size to sufficiently
resolve while at the same time still allowing lower mol-wt proteins to separate lower
down the gradient. To produce a concave gradient, place 7.5 mL of solution B in reservoir
B, then tightly stopper this reservoir with a rubber bung. Equalize the pressure in the
chamber by briefly inserting a syringe needle through the bung. Now place 22.5 mL of
solution A in reservoir A, open the connector between the two chambers, and commence
pouring the gel. The volume of reservoir B will be seen to remain constant as liquid for
reservoir A is drawn into this reservoir and diluted.