Analytical techniques

1. Iso Electric Focusing
By Dr Farhan Siddique

2. Introduction:
 Isoelectric focusing (IEF) is also known as electro
focusing.
 It is a technique for separating different molecules
by differences in their isoelectric point (pI).

3.  The isoelectric point (pI, pH(I), IEP),
is the pH at which a molecule
carries no net electrical charge or is
electrically neutral in the statistical
mean.
 The standard nomenclature to
represent the isoelectric point is
pH(I). However, pI is also used.

4.  It is a type of zone electrophoresis usually performed
on proteins in a gel that takes advantage of the fact
that overall charge on the molecule of interest is a
function of the pH of its surroundings.

6.  A protein that is in a pH region below its isoelectric
point (pI) will be positively charged and so will migrate
toward the cathode (negatively charged electrode).
 As it migrates through a gradient of increasing pH,
however, the protein's overall charge will decrease until
the protein reaches the pH region that corresponds to
its pI.

7.  At this point it has no net charge and so migration ceases (as
there is no electrical attraction toward either electrode).
 As a result, the proteins become focused into sharp stationary
bands with each protein positioned at a point in the pH
gradient corresponding to its pI.

8.  The technique is capable of extremely high
resolution with proteins differing by a single charge
being fractionated into separate bands

11.  Isoelectric focusing requires solid support such as
agarose gel and polyacrylamide gel
 Isoelectric focusing gels contain synthetic buffers called
ampholytes that smooth the pH gradients.
 Ampholytes are complex mixtures of synthetic
polyamino-polycarboxylic acids
Commercially available ampholytes are-
BIO-LYTE
PHARMALYTE