Isoelectric focusing electrophoresis Isoelectric-focusing electrophoresis is a type of electrophoresis. The separation technique involves electrophoresis based on the isoelectric point of the sample.

1. Isoelectric focusing
electrophoresis
Nishanth K P
1st Semester
M. Pharm (Pharmaceutics)
Nazareth College of Pharmacy 1

2. INTRODUCTION
IEF electrophoresis is began in 1964
In isoelectric Iso means ‘same’ and Electric means ‘charge’
Iso electric focusing (IEF), is a technique for separating different
molecules by based on their isoelectric point
The isoelectric point is the pH at which the net charge of the
protein is Zero.
IEF is often used as part of the quality control testing of
therapeutic biological products to demonstrate batch consistency.
2

3. PRINCIPLE
IEF is performed in a pH gradient
Proteins are amphoteric molecules with acidic and basic buffering
groups
In basic environment, the acidic groups become negatively charged
In acidic environment, the basic groups becomes positively charged
Isoelectric point (pI) : The pH where the charge of a protein is zero
Proteins with same molecular weights will separate out by pH
3

4. 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
4

5. All proteins have an isoelectric pH
When the electrophoresis is run in a solution buffered at constant pH,
Proteins having a net charge will migrate towards the opposite
electrode so long as the current flows.
The use of pH gradient across the supporting medium causes each
protein to migrate to an area of specific pH. The pH of the protein
equals the pH of the gradient, thus resulting in sharp well defined
protein bands.
A procedure to determine the isoelectric point (pI) of proteins thus, a
mixture of proteins can be electrophorized through a solution having a
stable pH gradient in from the anode to the cathode and a each protein
will migrate to the position in the pH gradient according to its
isoelectric point. This is called isoelectric focusing
IEF is also known as Electrofocusing 5

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

7. 7

8. Separation is achieved by applying a potential difference across a gel
that contains a pH gradient
Isoelectric focusing requires solid support such as agarose gel and
polyacrylamide gel
Isoelectric focusing gels contains synthetic buffers called ampholytes
that smooth the pH gradients
Ampholytes are complex mixtures of synthetic poly amino poly
carboxylic acids
Commercially available ampholytes are;
-BIO-LYTE
-PHARMALYTE
8

9. Advantages
IEF, 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
9

10. APPLICATIONS
IEF is a highly sensitive analytical technique and is particularly useful
for studying microheterogeneity in a protein
The method is useful for separating and identification of isoenzymes
(Which are different forms of the same enzyme often differing by only
one or two amino acid residues)
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
Protein purification
To measure pI values
Immuno blotting assays
10

11. REFERENCES
 Friedman DB, Hoving S, Westermeier R. Isoelectric focusing and two-
dimensional gel electrophoresis. Methods in enzymology. 2009 Jan
1;463:515-40.
 https://www.excedr.com/resources/isoelectric-focusing-electrophoresis-
overview
 https://www.slideshare.net/arushe143/isoelectric-focussing
11

12. Thank
You
12