Isoelectric focusing is a technique that separates proteins and other amphoteric molecules based on their isoelectric point, which is the pH at which they have no net charge. During isoelectric focusing, proteins are placed in an immobilized pH gradient formed by carrier ampholytes and an electric field is applied, causing the proteins to migrate through the pH gradient until they reach the pH that matches their isoelectric point and stop migrating. It is useful for analyzing protein heterogeneity and separating isoforms that have small charge differences but similar molecular weights.

1. ISOELECTRIC
FOCUSSING

2. Introduction
Principle
Procedure
Applications
ISOELECTRIC FOCUSSING

3.  Isoelectric focusing (IEF), is a technique for separating different
amphoteric molecules by based on their isoelectric point.
 The isoelectric point is the pH at which the net charge of the
protein is zero.
 IEF began in 1964, when Olaf Vesterberg filed a Swedish patent
on the synthesis of new chemicals called carrier ampholytes.
 This technique was popularized by H. Svensson in Sweden.
Isoelectric
Iso: “same” Electric : “charge”
INTRODUCTION

4. • Proteins with same molecular weights will separate out by pH
 IEF is preformed in a pH gradient.
 Proteins are amphoteric molecules with acidic and basic
buffering groups (side chain).
• In basic environment, the acidic groups become negatively
charged.
• In acidic environment, the basic groups become positively
charged.
• Isoelectric point (pI): the pH where the charge of a protein is
zero.
PRINCIPLE

5. • Proteins are positively charged in
solutions at pH values below pI and
migrate towards cathode.
• Proteins are negatively charged in
solutions at pH values above pI and
migrate towards anode.
Cont…

6. • Protein is loaded at the top of a column where pH is very high.
• Most of them are negatively charged at this pH.
• Protons are stripped from residue side chains.
• Proteins move in the electric field toward the distant cathode and away
from the nearby anode.
• As the proteins move through the pH gradient, they gain positive charge
and reach neutrality.
• At pH=pI, the proteins have no charge and stop
Procedure

7. Proteins stop exactly when
pH=pI and the stained
proteins are very visible
Highly stable ampholytes are
molecules with specific pKa
to give a specific and
unchanging pH gradient

8. • Seperation is achieved by applying a potential difference across a
gel that contain a pH gradient
• 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

9. First developed by Righetti ,(1990).
Immobilized pH gradient generated by buffering acrylamide
derivatives
Immobilized pH gradient, IPG
The film-supported gel strips are easy to handle.
The fixed gradient are consistent during IEF.
Less protein loss than carrier ampholytes.

11. • IEF is a highly sensitive analytical technique and is particularly useful
for studying microheterogeneity in a protein.
For example: a protein may show a single band on an SDS gel, but may
show three bands on an IEF gel. This may occur, for example, when a
protein exists in mono-, di- and tri-phosphorylated forms. The difference
of a couple of phosphate groups has no significant effect on the overall
relative molecular mass of the protein, hence a single band on SDS gels,
but the small charge difference introduced on each molecule can be
detected by IEF.
• The method is useful for separating isoenzymes (which are different
forms of the same enzyme often differing by only one or two amino
acid residues)
Applications

12. • 2D Gel electrophoresis is an application of IEF. Protein is first
separated based on pI and then based on molecular weight using
SDS-PAGE.
• Widely used for separation and identification of serum proteins.
For example: Detection of oligoclonal bands in gamma-globulin with the
use of IEF.

13. • IEF's greatest advantage is its high resolution, resulting in greater
separation of solutes. IEF of serum proteins results in many more bands;
these bands are sharper because each pH region is very narrow.
• Performing IEF is easier because the placement of sample application is
not important. The sample and ampholytes can be mixed before
application; the ampholytes will migrate, create the gradient, and then the
proteins separate and migrate.
Advantages