Anode, Cathode, 3 isoelectric focusing – Bio-Rad Rotofor® and Mini Rotofor Cells User Manual

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1.3 Isoelectric Focusing

Isoelectric focusing (IEF) is a gentle, non-denaturing technique; antibodies,

antigens, and enzymes usually retain their biological activities. IEF is also a high
resolution technique capable of resolving proteins that differ in pI by fractions of a
pH unit. IEF in the Rotofor has the added advantage that the proteins can be easily
recovered once they are focused.

Separation of proteins by isoelectric focusing is based on the fact that all proteins

have a pH-dependent net charge. The net charge is determined both by the amino
acid sequence of the protein and the pH of the environment. When a protein is
electrophoresed through an established pH gradient, it will migrate until it reaches the
pH where the net charge on the protein is zero; at that point it will stop migrating and
is said to be focused at its isoelectric point or pI.

Ampholytes which are small, charged buffer molecules are used to establish

the pH gradients increasing in pH from anode to cathode. When voltage is applied
to a system of ampholytes and proteins, all the components migrate to their
respective pIs. Ampholytes rapidly establish the pH gradient and maintain it for
long periods allowing the slower moving proteins to focus.

A protein with a net positive charge, for example, in a particular region of the pH

gradient will tend to migrate toward the cathode while concurrently giving up protons.
At some point, the net charge on the molecule will be zero and the protein will cease
to migrate. If the protein diffuses into a region of net charge, the resultant electrical
force on it will drive it back to its pI, so that the molecule becomes focused at that
point.

Fig. 1.1. Acidic Protein “Focusing” in a pH gradient.

Anode

(+)

Cathode

(-)

net charge

(+2)

3 4 5 6 7 8 9

(0)

(-2)

COOH

COO