Isoelectric Focusing (IEF)
IEF is an electrophoretic method for separating proteins based on
their isoelectric point. The isoelectric point is the pH at which
the net charge of the protein is zero. With the presence of a pH
gradient in the IEF technique, the protein will migrate to the
position in the gradient where its charge is zero. Proteins with a
positive net charge will migrate toward the cathode until it meets
its pI. Proteins with a negative net charge will migrate toward the
anode until it meets its pI. If the protein diffuses away from its
pI, it will regain its charge and migrate back. This focusing effect
allows proteins to be separated based on very small charge
differences. IEF is performed under high voltages (> 1000 V)
until the proteins have reached their final position in the pH
gradient. If IEF is performed under denaturing conditions very high
resolution and cleanliness of sample can be obtained.
IEF is performed using the IPGphor Isoelectric Focusing System from
Amersham Pharmacia Biotech.
This system uses immobilized pH gradient (IPG) gels with a plastic
backer. These gels increase reproducibility, loading capacity and
are easier to handle. The IPG strips are available in either 7cm
(for mini-gel application) or 18cm (for larger gel applications)
sizes.
- 2-D/IEF Submission Form (PDF)
