Preparation of the Gels
Composed of a glass plate (A) on which a polyester film (B) is placed to facilitate handling of the gel, one or more spacers (C), a second glass plate (D), and clamps to hold the structure together (see Figure 1
7.5% Polyacrylamide Gel
Dissolve 29.1 g of acrylamide and 0.9 g of methylenebisacrylamide in 100 mL of water. To 2.5 volumes of this solution, add the mixture of ampholytes specified in the individual monograph, and make up to 10 volumes with water. Mix carefully, and degas the solution.
Preparation of the Assembly
Place the polyester film on the lower glass plate, apply the spacer, place the second glass plate, and fit the clamps. Before use, place the mixture on a magnetic stirrer, and add 0.25 volumes of a 10% solution of ammonium persulfate and 0.25 volumes of tetramethylenediamine. Immediately fill the space between the glass plates of the assembly with the gel.
Fixing Solution for Isoelectric Focusing Polyacrylamide Gel
Mix 35 g of sulfosalicylic acid and 100 g of trichloroacetic acid in 1000 mL of water.
Coomassie Staining Solution and Destaining Solution
Use the same solutions indicated in Polyacrylamide Gel Electrophoresis.
Dismantle the assembly, and using the polyester film, transfer the gel onto the cooled support wetted with a few milliliters of a suitable liquid, taking care to avoid forming air bubbles. Prepare the test solutions and reference solutions as specified in the individual monograph. Place strips of paper for sample application, about 10 mm × 5 mm in size, on the gel, and impregnate each with the prescribed amount of the test and reference solutions. If the protein concentration of the solution is too low, several strips may be superimposed (up to four). Also apply the prescribed quantity of a solution of proteins with known isoelectric points as pH markers to calibrate the gel. In some procedures, the gel has pre-cast slots where a solution of the sample is applied instead of using impregnated paper strips. Cut two strips of paper to the length of the gel, and impregnate them with the electrolyte solutions: acid for the anode and alkaline for the cathode. The compositions of the anode and cathode solutions are given in the individual monograph. Apply these paper wicks to each side of the gel several millimeters from the edge. Fit the cover so that the electrodes are in contact with the wicks (with respect to the anodic and cathodic poles). Proceed with the isoelectric focusing by applying the electrical parameters described in the individual monograph. Switch off the current when the migration of the mixture of standard proteins has stabilized. Using forceps, remove the sample application strips and the two electrode wicks. Immerse the gel in Fixing Solution for Isoelectric Focusing Polyacrylamide Gel. Incubate with gentle shaking at room temperature for 30 minutes. Drain off the solution, and add 200 mL of Destaining Solution. Incubate with shaking for 1 hour. Drain the gel, add Coomassie Staining Solution. Incubate for 30 minutes. Destain the gel by passive diffusion with Destaining Solution until the bands are well visualized against a clear background. Locate the position and intensity of the bands in the electropherogram as prescribed in the individual monograph.
When a monograph references the general method for isoelectric focusing above, variations in methodology or procedure may be used, subject to validation. These variations include the use of commercially available pre-cast gels; the use of immobilized pH gradients; the use of rod gels; and the use of cassettes of different dimensions, including ultra-thin (0.2 mm) gels; the variations in the sample application procedure, including different sample volumes or the use of sample application masks or wicks other than paper; the use of alternate running conditions, including variations in the electric field depending on gel dimensions and equipment, and the use of fixed migration times rather than subjective interpretation of band stability; the inclusion of a pre-focusing step; the use of automated instrumentation; and the use of agarose gels.