Use of a DNA Sequencing Gel Apparatus for Analysis of Polypeptides

Polyacrylamide gel electrophoresis (PAGE) is a standard procedure for identifying and analyzing polypeptides (1). Samples are prepared from cell lysates, subjected to electrophoresis and stained with silver nitrate or Coomassie Brilliant Blue (CBB) allowing direct visualization of bands within gels (1,5). Samples can also be radiolabeled, for example with [35S]methionine, and after electrophoresis, the resulting gels are dried on chromatography paper and autoradiographed (3). A wide variety of electrophoresis chambers are available for producing gels for these types of analyses. We describe here a procedure, which allows one to use a nucleic acid sequencing gel apparatus instead of the conventional equipment. The main advantage of this method is that it enables the use of one apparatus for both DNA sequencing and polypeptide work. To prepare a sequencing apparatus before sealing, we used a Sequi-Gen cell (21× 50-cm) (Bio-Rad, Hercules, CA, USA) for carrying out this procedure. The clamp set, which is used to hold the glass plates together, should be in good condition. Warped or uneven clamps cause leakage at the sides during gel pouring. Plates and spacers are washed normally and rinsed with 70% ethanol. No treatment is required to selectively bind the acrylamide gel to the outer glass plate. Four 0.4-mm spacers are required. These are doubled together along each side of the apparatus, giving a gap of 0.8 mm, once the assembly is clamped together. Thinner gels (0.4 mm) are extremely difficult to pour, and they break easily at the plate separation stage. The gap generated by the double set of spacers does not allow for the use of standard 0.8-mm combs for protein gel combs. This is overcome by using the Bio-Rad 14-cm plastic square combs (16 well), which are designed for sequencing. Two combs of 0.4-mm thickness are glued (or can be taped) together to form a single comb, which fits well in the gap made by the spacers. If combs are glued, a high-strength adhesive should be used so as not to increase the width significantly, and the edges of the comb are trimmed with a scalpel after gluing. New combs should be tested before pouring gels. The only other extra requirement is approximately 10 cm3 of modeling clay or plasticine, which is used for sealing the bottom of the assembled apparatus. The plasticine should be kneaded before use to make it very malleable and is split into three lots, two of 0.5 cm3 each and the remainder. The smaller pieces are pressed at the bottom corners of the assembly, over the areas where the ends of the spacers are located. Care should be taken to ensure that this area is completely dry as moisture can inhibit the sealing capacity of the plasticine plugs.