Isoelectric Point (pI)
Definition
pH at which a molecule (usually a protein) carries zero net charge and stops migrating. The basis of isoelectric focusing (IEF).
- Defines
- pH of zero net protein charge
- Underlies
- Isoelectric focusing (IEF)
- Behaviour at pI
- Protein stops migrating in an electric field
- Applied to
- Serum and enzyme proteins in forensic serology
Common questions
How does pI actually separate proteins during IEF?+
A gel is cast with a stable pH gradient. Under an applied voltage each protein migrates until it reaches the point in the gradient matching its own pI, where its net charge is zero, then it stops moving. Proteins with different pI values end up focused at different positions.
Why is pI useful when DNA typing is not an option?+
Before STR typing became standard, isoenzyme and serum protein phenotypes separated by pI gave a way to narrow down the possible donors of a bloodstain. It is now mainly of historical and cold-case interest but remains relevant to understanding older case files.
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...
- IEF (Isoelectric Focusing)
- Electrophoresis across an immobilised pH gradient. Each protein migrates until it reaches its pI and stops. Very high resolution; used in haemoglobin...
- Immunoelectrophoresis
- A two-step technique: proteins are first separated by electrophoresis in agar gel, then antiserum diffuses from a parallel trough and forms precipitin...
- 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.