IEF involves adding an
ampholyte solution into
immobilized pH gradient (IPG) gels. IPGs are the
acrylamide gel matrix co-polymerized with the pH gradient, which result in completely stable gradients except the most alkaline (>12) pH values. The immobilized pH gradient is obtained by the continuous change in the ratio of
immobilines. An immobiline is a weak acid or base defined by its pK value. A protein that is in a pH region below its
isoelectric point (pI) will be positively charged and so will migrate toward the cathode (negatively charged electrode). As it migrates through a gradient of increasing pH, however, the protein's overall charge will decrease until the protein reaches the pH region that corresponds to its pI. At this point it has no net charge and so migration ceases (as there is no electrical attraction toward either electrode). As a result, the proteins become focused into sharp stationary bands with each protein positioned at a point in the pH gradient corresponding to its pI. The technique is capable of extremely high resolution with proteins differing by a single charge being fractionated into separate bands. Molecules to be focused are distributed over a medium that has a pH gradient (usually created by
aliphatic ampholytes). An
electric current is passed through the medium, creating a "positive"
anode and "negative"
cathode end. Negatively charged molecules migrate through the pH gradient in the medium toward the "positive" end while positively charged molecules move toward the "negative" end. As a particle moves toward the pole opposite of its charge it moves through the changing pH gradient until it reaches a point in which the pH of that molecule's isoelectric point is reached. At this point the molecule no longer has a net electric charge (due to the protonation or deprotonation of the associated functional groups) and as such will not proceed any further within the gel. The gradient is established before adding the particles of interest by first subjecting a solution of small molecules such as
polyampholytes with varying pI values to electrophoresis. The method is applied particularly often in the study of
proteins, which separate based on their relative content of
acidic and
basic residues, whose value is represented by the pI. Proteins are introduced into an
immobilized pH gradient gel composed of
polyacrylamide,
starch, or
agarose where a pH gradient has been established. Gels with large pores are usually used in this process to eliminate any "sieving" effects, or artifacts in the pI caused by differing migration rates for proteins of differing sizes. Isoelectric focusing can resolve proteins that differ in
pI value by as little as 0.01. Isoelectric focusing is the first step in
two-dimensional gel electrophoresis, in which proteins are first separated by their pI value and then further separated by
molecular weight through
SDS-PAGE. Isoelectric focusing, on the other hand, is the only step in preparative native PAGE at constant pH. ==Living cells==