Friday, January 8, 2010

What is a pH gradient in electrophoresis?

I've read that it occurs when molecules move towards the anode and cathode and some stay in between, but i still dont get what it is.What is a pH gradient in electrophoresis?
When you are doing a typical electrophoresis you use a buffer and make the gel so that the pH is uniform everywhere in the gel.





If you want to do electrophoresis with a pH gradient (isoelectric focusing) then you need to include in your gel special compounds that will make the gel to have areas with different pH values. These are not random but are positioned along the axis of the direction of the movement of the proteins and gradually increase or decrease.





What's the point?


A protein has many groups that can be positively charged and many that can be negatively charged. The direction of its movement depends on the net charge of the protein. If it is positive it will move towards the cathode, if it is negative it will move towards the anode and if it is zero it will not move.





The net charge depends on the pH. So some proteins at a given pH value will have a net charge and move within the gel because of the applied electric field. When they reach a different area of the gel with different pH their net charge will also change and thus their movement will be affected. At a certain pH value (called the isoelectric point pI) the net charge of a protein is 0. Then the electric field will not exert a force on this protein anymore and it will stop at that position.


Each protein has different amino acid composition and thus each protein has its own pI value.

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