IEF (Isoelectric Focusing)
Definition
Electrophoresis across an immobilised pH gradient. Each protein migrates until it reaches its pI and stops. Very high resolution; used in haemoglobin variant typing.
- Method type
- Electrophoresis on an immobilised pH gradient
- Migration endpoint
- Each protein stops at its isoelectric point (pI)
- Resolution
- Very high, resolves proteins differing by small pI
- Forensic use
- Haemoglobin variant typing
Common questions
Why does a protein stop moving once it reaches its isoelectric point?+
At its pI the protein carries no net electric charge, so the electric field no longer pulls it in either direction along the gradient. It effectively self-focuses at that position, which is why the technique achieves sharper bands than standard gel electrophoresis.
Why is IEF particularly useful for haemoglobin variant typing compared to ordinary electrophoresis?+
Many haemoglobin variants differ from normal haemoglobin by a single amino acid substitution that produces only a small charge difference. IEF's high resolving power on a pH gradient can separate these closely related variants that standard gel methods would run together as one band.
Related terms
- Agarose
- Polysaccharide gel from seaweed. Large pore size, used for nucleic acids and large proteins. Standard for DNA submarine gels.
- Capillary Electrophoresis (CE)
- A separation technique that resolves DNA fragments by size through a polymer-filled glass capillary under an applied electric field. Single-base resolution is...
- Electrophoretic Mobility (μ)
- Velocity of a charged particle per unit electric field, μ = v/E. Depends on charge, size, shape and the viscosity of the...
- Immunoelectrophoresis
- A two-step technique: proteins are first separated by electrophoresis in agar gel, then antiserum diffuses from a parallel trough and forms precipitin...
- Isoelectric Point (pI)
- pH at which a molecule (usually a protein) carries zero net charge and stops migrating. The basis of isoelectric focusing (IEF).
- Polyacrylamide (PAGE)
- Cross-linked synthetic gel with tunable, smaller pore size. Used for proteins and small DNA fragments.
- Precipitin Arc
- Curved line of antigen-antibody precipitate that forms at equivalence in immunoelectrophoresis. Shape and position identify the antigen.
- SDS-PAGE
- PAGE run with sodium dodecyl sulphate. SDS coats proteins with uniform negative charge, so separation is by molecular weight alone.