Bio-Rad Model 111 Mini IEF Cell - Manual (Page 8)

Section 2
Introduction to Isoelectric Focusing
2.1 The Electrofocusing Principle
Conventional electrophoresis separates proteins and other charged molecules by
electrophoretically-driven migration through a sieving matrix that is buffered at a constant pH.
Each component of the mixture assumes its own characteristic velocity based on molecular size
and surface charge. This velocity is constant throughout the electrophoresis experiment and is
counteracted by diffusion, which tends to broaden the bands. There is no tendency toward
equilibrium in conventional electrophoresis, and the protein bands will run off the gel if the
electrical field is not interrupted.
On the other hand, electrofocusing separates proteins on the basis of surface charge alone as a
function of pH. The separation is done in a non-sieving medium (sucrose density gradient,
agarose, or polyacrylamide gel) in the presence of carrier ampholytes, which establish a pH
gradient increasing from the anode to the cathode. Since a protein contains both positive (amines)
and negative (carboxyl) charge-bearing groups, the net charge of the protein will vary as a
function of pH.
A pH gradient is established concomitantly with protein separation. As the protein migrates
into an acidic region of the gel, it will gain positive charge via protonation of the carboxylic and
amino groups. At some point, the overall positive charge will cause the protein to migrate away
from the anode (+) to a more basic region of the gel. As the protein enters a more basic
environment, it will lose positive charge and gain negative charge, via ammonium and carboxylic
acid group deprotonation, and consequently, will migrate away from the cathode (-). Eventually,
the protein reaches a position in the pH gradient where its net charge is zero (defined as its pI or
isoelectric point). At that point, the electrophoretic mobility is zero. Migration will cease, and a
concentration equilibrium of the focused protein is established.
2.2 Carrier Ampholytes
Carrier ampholytes are complex mixtures of amphoteric buffers that form a smooth pH
gradient in an applied electrical field. During electrofocusing, these buffers stack according to
their individual pIs across the gel, producing a linear gradient. In order for the gradient to appear
smooth and continuous, a large number of these buffering components must be present. This is
also a requirement for separating a complex mixture of proteins.
Bio-Lyte ampholytes are derivatized low-molecular weight amines that are electrophoretically
separated and reblended to give smooth and reproducible gradients. Narrow range Bio-Lyte
ampholytes are produced and tested so that, under normal circumstances, no additional blending
or fortification will be necessary to achieve the desired shallow gradient.
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