Isoelectric focusing is a technique that separates molecules like proteins based on their isoelectric point, which is the pH at which the molecule has no net charge. It was developed in the 1960s and allows for much better resolution than older techniques. The process involves creating an immobilized pH gradient using carrier ampholytes, loading protein samples, and applying an electric field to cause proteins to migrate to the point in the gradient matching their isoelectric point. The separated proteins can then be visualized through staining. Isoelectric focusing is useful for applications like identifying serum proteins and aiding in proteomics research.

1. ISOELECTRIC FOCUSING
•PAUL SINGH

2. ISOELECTRIC FOCUSING
 Isoelectric 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 also known as electrofocusing,

3. HISTORY

4. HISTORY
 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.
 Highly efficient.
 If paper electrophoresis resolves plasma proteins into six
bands, Isoelectric focussing resolves it into at least 40
bands.

5. CARRIER AMPHOLYTE

6. CARRIER AMPHOLYTE
 Ampholytes are low molecular weight molecules that
help in creating a stable pH gradient.
 They are isomers of polycarboxylic acids.
 PROPERTIES:-
 It should be soluble in water.
 It should have low absorption spectra.
 It should behave as a behave and offer
conductance.

7. BASICS OF ISOELECTRIC
FOCUSING

8.  If the number of acidic groups in a protein exceeds
the number of basic groups, the pI of that protein will
be at a low pH value and the protein is classified as
acidic.
 Similarly if the number of basic groups in a protein
exceeds the number of acidic groups, the pI of that
protein will be at a high pH an the protein is
classified as basic.
 Proteins show a pI value of 4-7 with the pH falling in
the range of 3-12.

9.  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.

10. PROCEDURE

11. PREPARATION OF GEL
 CHEMICALS REQUIRED:-
ACRYLAMIDE, AMMONIUM PERSULPHATE(APS),
TEMED, CARRIER AMPHOLYTE, UREA, WATER
 Required amount of acrylamide is dissolved in distilled water.
 Remove air bubbles if present.
 Add solution containing of carrier ampholytes to the mixture.
 Mix thoroughly
 Add 10%APS and TEMED
 Pour the mixture into the gel cassette.
 Place the comb in the gel cassette
 Allow the gel to solidify
 Remove comb after solidification.

12. SET UP OF THE APPARATUS
 Connect the terminals of the power supplier to
electrodes of the gel chamber. (Black= Cathode ;
Red= Anode)
 Pour catholyte (Sodium hydroxide) in the upper
buffer chamber and Anolyte (Phosphoric acid) in the
lower buffer chamber

13. SAMPLE PREPARATION AND LOADING
 Protein sample is mixed with a equal amount of gel
loading buffer.
 Mix the sample and the buffer thoroughly.
 Load the sample onto the wells.
 After loading, run the gel for 30 mins at 150V and for
2 hours at 200V.
 Samples are separated based on their isoelectric
points.

14. CALCULATING pH
 After the time period of electrophoresis, the power is
turned off and the gel is blot dried.
 The band containing protein sample is cut into
pieces and the distance of the band from any one of
the electrode is calculated.
 The band is then incubated in a solution of
Potassium chloride, by doing this pH of the band is
obtained.
 A graph is plotted taking pH of the band on X-Axis
and distance of band on Y-Axis.
 A standard curve is obtained and pI value is
calculated.

15. FIXING AND STAINING OF GEL
 Soak the gel in 10%TCA for removal of ampholytes
 Ampholytes are removed to avoid background
staining
 Stain the gel using coomasie blue for 10min.
 Discard staining solution and place the gel in
destaining solution.

16. APPLICATIONS

17. APPLICATIONS
 Widely used for separation and identification of
serum proteins.
 Used in food and agricultural industries, forensic and
human genetics laboratories.
 Used in enzymology, immunology and membrane
biochemistry.
 2D Gel electrophoresis is an application of IEF.
Protein is first separated based on pI and then based
on molecular weight using SDS-PAGE.