First pour the separating gel, overlay water on the surface to eliminate air bubbles, and pour off the water layer after solidification.

Fill up with stacking gel and insert the comb; pull out the comb gently once fully polymerized. Prepare gel solutions freshly before use; store APS and TEMED away from light at low temperature.

Avoid air bubbles during pouring, clamp gel plates tightly to prevent leakage. Select gel concentration according to target protein molecular weight.

Let the gel polymerize undisturbed at room temperature without shaking. Pull the comb out steadily to keep sample wells intact and straight.