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The isoelectric point of most proteins is between pH 4.0 and 7.5. In pH 8-9, proteins will take on a negative charge and migrate to the anode. Most protein electrophoresis is performed at pH 8.6.

Buffers most commonly used are barbital or tris-boric acid-EDTA buffers. They fix the pH at 8.6, leading to sharper bands and good separations.