Dismantle the Mold, and using the polyester film, transfer the gel onto the cooled support wetted with a few mL 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. 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 precast 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 mm 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, and 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.

Where reference to the general method on isoelectric focusing is made, variations in methodology or procedure may be made subject to validation. These variations include the use of commercially available precast gels and of commercial staining and destaining kits; 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; 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 prefocusing step; the use of automated instrumentation; and the use of agarose gels.