Isoelectric focusing, its history and how it is performed.

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.