isoelectric focusing (IEF)
Uses
- serum acid phosphatase isoenzymes
- oligoclonal immunoglobulin bands in CSF
- serum creatine kinase isoenzymes
- serum alkaline phosphatase isoenzymes
Procedure
IEF is performed similarly to other electrophoretic methods except that the separating molecules migrate through a pH gradient. Proteins (& other molecules) cease migration at their isoelectric pH (pI).
The pH gradient is created by addition of acid to the anodic area of the electrolytic cell & base to the cathodic area. A solution of ampholytes with different pI is placed between the 2 electrodes. These ampholytes have high buffering capacity at their respective pI. When an electric field is applied, each ampholyte will rapidly migrate to its pI. With their high buffering capacity, the ampholytes create stable pH zones for more slowly migrating proteins or other macromolecules.
Using a narrow range of ampholytes, macromolecules differing in pI of only 0.02 may be resolved.
More general terms
Additional terms
References
- ↑ Clinical Diagnosis & Management by Laboratory Methods, 19th edition, J.B. Henry (ed), W.B. Saunders Co., Philadelphia, PA. 1996, pg 65