Ion Focusing, SDS, and Two-Dimensional Electrophoresis

In Chapter 27, p. 322, you learned how a molecule can be neutral or negatively or positively charged, depending upon the pH of the solution it is in. 

There are two main techniques for establishing a pH gradient along a strip nongel and gel. Both require compounds that have a variable charge but will become neutral at a certain pH. These are called ampholytes. 

When you studied the Periodic Table, you learned about amphoteric elements, those that would act like a base in some cases and like an acid in others. When a molecule has that ability and is used in an electrophoresis separation, it is called an ampholyte. Ampholytes are used as pH markers and to establish the pH gradient. There are two basic systems, natural and immobilized. 

Available from Pharmacia Biotech as Immobiline, these are derivatives of acrylamide with different dissociation constants. For acidic Immobiline (with pK 3.6 or 4.6), carboxylic acid is chosen as a functional group for the bases, tertiary amino groups are selected. When different pK values of Immobiline are mixed in appropriate proportions, together with acrylamide, bisacrylamide, TEMED, and ammonium persulfate, the buffering groups responsible for ereating the pH gradient become covalently attached to the polyacrylamide base. 

The pH of the gel can be determined by inserting a microelectrode at points along the gel, or a small section can be cut out and dissolved, and the pH measured with a regular set of electrodes. A common set of microelectrodes includes antimony wire for the indicator electrode and a micro calomel electrode for the reference. If you use this pair, you must correct the pH meter reading based upon the half cell reaction. 

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Overall, electrophoresis is to ions what chromatography is to neutral molecules. There is one report of a two-dimensional separation involving ion focusing and SDS to resolve over 1000 compounds on a single sheet. That is amazing. 

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