Isoelectric Focusing and 2D gel electrophoresis

Iso-Electric focusing is widely used for proteome analysis. Isoelectric focusing is based on the concept of isoelectric pH. The pH at which net charge on protein becomes zero, is called isoelectric pH of protein. Such protein will have equal number of positive and negative charge. As a result net charge become zero. The separation of proteins is carried out on gel with pH gradient. Isoelectric focusing is often followed by electrophoresis in second dimension. This is also known as Two dimensional gel electrophoresis (2D gel electrophoresis)

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[@tonytong6682]

if there is no pH gradient in a gel, does high pI value still means it travels further to the anode? since anode is positive, and high pI value means it is more basic so it is more attracted to the anode than lower pI proteins?

[@iglaniket1352]

Due to presence of both negative and positive charge on protein .protein runs until it's net charge become 0 .in which pH it becomes 0 is called it's isoelectric pH. If protein is in high pH(suppose 11) than its iso electric pH(suppose 8) it will run backword on pH strip(ipg strips) when it's provide electric charge as it will carry negative charge and run to positive charge {vice versa for protein which is in lower pH than its iso electric pH} so both will run until their net charge becomes 0 so we can seperate on this basis &then run the same process as SDS - PAGE.
(Ignore My Grammer as I am not a native English speaker)