Protein Protocols Protein Protocols

70 Walker
2. Buffers:
a. 1.875 M Tris-HCl, pH 8.8.
b. 0.6 M Tris-HCl, pH 6.8.
Store at 4°C.
3. Ammonium persulfate solution (10% [w/v]). Make fresh as required.
4. SDS solution (10% [w/v]). Stable at room temperature. In cold conditions, the SDS can
come out of solution, but may be redissolved by warming.
5. N,N,N',N'-Tetramethylene diamine (TEMED).
6. Gradient forming apparatus (see Fig. 1). Reservoirs with dimensions of 2.5 cm id and
5.0 cm height are suitable. The two reservoirs of the gradient former should be linked by
flexible tubing to allow them to be moved independently. This is necessary since although
equal volumes are placed in each reservoir, the solutions differ in their densities and the
relative positions of A and B have to be adjusted to balance the two solutions when the
connecting clamp is opened (see Note 3).
3. Method
1. Prepare the following solutions:
Solution A, mL Solution B, mL
1.875 M Tris-HCl, pH 8.8 3.0 3.0
Water 9.3 0.6
Stock acrylamide, 30% 2.5 10.0
10% SDS 0.15 0.15
Ammonium persulfate (10%) 0.05 0.05
Sucrose — 2.2 g
(equivalent to
1.2 mL volume)
2. Degas each solution under vacuum for about 30 s and then, when you are ready to form the
gradient, add TEMED (12 µL) to each solution.
3. Once the TEMED is added and mixed in, pour solutions A and B into the appropriate
reservoirs (see Fig. 1.)
4. With the stirrer stirring, fractionally open the connection between A and B and adjust the
relative heights of A and B such that there is no flow of liquid between the two reservoirs
(easily seen because of the difference in densities). Do not worry if there is some mixing
between reservoirs—this is inevitable.
5. When the levels are balanced, completely open the connection between A and B, turn the
pump on, and fill the gel apparatus by running the gel solution down one edge of the gel
slab. Surprisingly, very little mixing within the gradient occurs using this method. A pump
speed of about 5 mL/min is suitable. If a pump is not available, the gradient may be run
into the gel under gravity.
6. When the level of the gel reaches about 3 cm from the top of the gel slab, connect the
pump to distilled water, reduce pump speed, and overlay the gel with 2–3 mm of water.
7. The gradient gel is now left to set for 30 min. Remember to rinse out the gradient former
before the remaining gel solution sets in it.
8. When the separating gel has set, prepare a stacking gel by mixing the following:
a. 1.0 mL 0.6 M Tris-HCl, pH 6.8;
b. 1.35 mL Stock acrylamide;
c. 7.5 mL Water;
d. 0.1 mL 10% SDS;
e. 0.05 mL Ammonium persulfate (10%).