High-temperature sodium dodecyl sulfate polyacrylamide gel electrophoresis.

The run time for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) can be reduced to as little as 5 min by running vertical slab gels at 70°C. High-temperature SDS-PAGE increases the migration rate of protein bands several fold and is as effective as cooling in preventing uneven migration rates across the width of the gel. Additional benefits of hightemperature SDS-PAGE include enhanced solubilization of samples, increased solubility of potassium salts and accelerated cross-linking of acrylamide gels. Tiselius (4) first recognized the benefit of cooling to prevent convective mixing in U tubes during electrophoretic runs. With the discovery of gel electrophoresis, convective mixing of the resolving matrix was no longer a problem. Nonetheless, gels are still routinely cooled during electrophoresis for other reasons. With nondenaturing gel electrophoresis, cooling reduces the denaturation of proteins. With denaturing gel electrophoresis in 8 M urea, cooling is necessary to reduce carbamylation of proteins. Although SDS-PAGE has no inherent requirement for cooling, cooling does prevent “smiling gels”, where the protein bands in the center lanes migrate faster than those in the outer lanes. The presumption is that proteins in the center lanes migrate faster because the temperature at the center of the gel is higher than at the edges because of uneven heat dissipation and/or uneven current density. Cooling the gel and running gels at lower voltage minimizes this temperature gradient, resulting in a more uniform migration rate. Unfortunately, cooling and low voltage also slow the rate of protein migration and can increase electrophoresis times to several hours. To some extent, gel electrophoresis times can be shortened by using high voltages and very thin gels with efficient cooling devices. Another popular approach has been the use of minigels, where the run time is shortened by simply reducing the distance the proteins migrate. In theory, heating the gel in a water bath during electrophoresis should prevent smiling and at the same time accelerate the migration of proteins. This follows from the assumption that it is the temperature gradient between the middle and the edges of the gel, relative to the absolute gel temperature, that determines the extent of smiling. For example, a temperature differential of 5°C between the middle and the edges of a gel would be significant if the gel temperature were 5°–10°C but would be less significant if the gel temperature were 65°–70°C. High temperatures per se are not a problem with SDS-PAGE, and samples are frequently heated at 100°C for 1–5 min in the presence of a reducing agent (e.g., dithiothreitol [DTT]) prior to electrophoresis. To test the effects of high temperature, we ran 8% SDS polyacrylamide gels at 70°C in a Hoefer SE 600 gel apparatus (Pharmacia Biotech, Piscataway, NJ, USA), using a slightly modified version of the buffer system described by Porzio and Pearson (3). This modification consisted of the following specifications: gel concentration 8% T, 5% C; running buffer: 200 mM Tris-glycine (50 mM Tris, 150 mM glycine), 0.1% SDS; gel buffer: 400 mM Tris-glycine (100 mM Tris, 300 mM glycine), 0.1% SDS, 5% glycerol, 0.2% polyacrylamide (2), 0.5 mM NaN3; sample buffer: 62.5 mM Tris base, 3% SDS, 20% glycerol, 6 mg/mL DTT, trace bromophenol blue, mixed 1:1 (vol/vol) with sample. Uniform heating of the gel was accomplished by completely submerging the gel in the lower (anodal) buffer preheated to 70°C. Figure 1 (A and B) illustrates the nearly identical resolution of molecular weight standards after 20 min at 69°C compared to 2.5 h at 12°C. Both gels were run at 500 V until the dye marker reached the bottom of the gel (13 cm). The mobility of the protein standards at 70°C was increased proportionately less than that of the dye front, and consequently, the protein bands in Panel B migrated about half as far as those in Panel A. The most notable feature of the gel in Panel B is that, like Panel A, the proteins in all the lanes have similar mobilities (i.e., no smile). Electrophoresis time could be further decreased by using minigels. To simulate the minigel format, 13-cm gels were run until the dye marker had migrated about 6 cm (Figure 1C). The total run time at 70°C was only 5 min. This compares to a 20–25-min run time required for the proteins to migrate an equal distance in a gel cooled to 10°C. There was very little loss in resolution under these conditions, as shown by the accompanying gel scan, and all nine protein standards were visibly resolved as individual bands. In addition to reducing run times by 75%–80%, high temperature has several other benefits, such as: (i) the rate of acrylamide cross-linking is greatly accelerated. Assuming a Q10 of 2, 10 min at 70°C is equivalent to 5.5 h at room temperature (20°C). A gel can be transferred to the 70°C buffer as soon as the gel becomes rigid enough to remove from the casting stand, and in the time it takes to load the samples into the wells (5–10 min), the cross-linking reaction should be nearly complete; (ii) SDS (1.5%, pH 8.8) is soluble with up to 700 mM KCl at 70°C compared to 10–15 mM at 10°C. Consequently, high-temperature SDS-PAGE allows for samples, such as ion-exchange column fractions (KCl gradient), to be run without prior removal of KCl. Of course, higher salt concentrations will distort the protein bands; (iii) the migration of the dye and proteins into the gel is significantly more uniform at high temperature, presumably because of the reduction in convective mixing in the sample well. The gels shown in Figure 1 were run with a continuous buffer system and without low-percentage acrylamide stacking gels. The samples shown in Panel A (12°C) were loaded into the gel at 100 V for 15 min, and then the gel was run at 500 V for 2.5 h. The samples shown in Panels B and C (69°–74°C) were run into the gels at 500 V, which was the same voltage used for the gel run. At lower temperatures, loading the proteins into the gel at such a high voltage would produce very uneven loading of the protein into the gel with the gel system used for these studies. By eliminating the lowpercentage acrylamide stacking gel and using high voltage for the entire