Technique combining ideas of isoelectric points and electric fields. It gives good separation with a high resolution compared to any other method. Proteins are separated in a pH gradient according to their isoelectric points, with proteins migrating towards the cathode or anode depending on whether their pI is below or above the pH at that point in the gradient. Applications include separation and identification of serum proteins in clinical settings and separation of proteins, enzymes, and other biomolecules in research.

1. I S F COLLEGE OF PHARMACY
MOGA
ISOELECTRIC FOCUSING
PRESENTED BY:
LOVEINDER BHARDWAJ
(PHARMACOLOGY)

2. Content
• INTRODUCTION
• ISOELECTRIC POINT
• PRINCIPLE
• AMPHOLYTE
• PROCEDURE
• SAMPLE PREPARATION AND LOADING
• CALCULATING PH
• APPLICATIONS

3. INTRODUCTION
• Isoelectric focusing (IEF), is a technique for separating different molecules by
based on their isoelectric point.
• It is also known as electro focusing.
• IEF is performed in a ph gradient.
Basic principle involved is electrophoresis
• IEF is well established as an excellent technique for the analysis of proteins,
such as enzymes, hormones or other biologically active proteins.

4. ISOELECTRIC POINT
• The ph. at which net charge on protein becomes zero.
• Proteins are separated in a pH gradient according to their isoelectric points.
• Proteins are amphoteric molecules with acidic and basic buffering groups (side
chain).
• 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.

6. PRINCIPLE
• Separates proteins by their isoelectric points.
• Each protein has own pl=ph at which the protein has equal amount of positive and negative
charges i.e the net charge is zero.

7. 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 offer conductance.

8. Synthetic carrier ampholyte:
 High buffering capacity and solubility at the pI.
 Good and regular electric conductivity at the pI.
 Absence of biological effects.
 Low molecular weight.
Natural occurring ampholyte:
 Amino acids or peptides
 Lack the properties above
 Can not be used in IEF.

9. What is a gel ?
 Gel is a cross linked polymer whose composition and porosity is chosen based
on the specific weight and porosity of the target molecules.
Types of Gel:
• Agarose gel.
• Polyacrylamide gel.

10. AGAROSE GEL
• A highly purified uncharged polysaccharide derived from agar.
• Used to separate macromolecules such as nucleic acids, large proteins and protein
complexes.
• It is prepared by dissolving 0.5% agarose in boiling water and allowing it to cool to 40°C.
• It is fragile because of the formation of weak hydrogen bonds and hydrophobic bonds.
• Nontoxic gel medium
• Good for separating large DNA molecules and poor for separating small DNA molecules.

11. POLYACRYLAMIDE GEL
• Used to separate most proteins and small oligonucleotides because of
the presence of small pores.
• Polyacrylamide gels are tougher than agarose gels.
• Polyacrylamide gels are composed of chains of polymerized
acrylamide.
• Sharp bands.

14. PREPARATION AND SAMPLING
• ACRYLAMIDE, AMMONIUM PERSULPHATE(APS), TEMED, CARRIER AMPHOLYTE, UREA,
WATER.
• An electrophoresis chamber and power supply
• Gel casting trays, which are available in a variety of sizes and composed of UV transparent plastic. The
open ends of the trays are closed with tape while the gel is being cast, then removed prior to electrophoresis.
• Sample combs, around which molten medium is poured to form sample wells in the gel.
• Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE).
• Loading buffer, which contains something dense (e.g. glycerol) to allow the sample to "fall" into the
sample wells, and one or two tracking dyes, which migrate in the gel and allow visual monitoring or how far
the electrophoresis has proceeded.

15. • Staining: DNA molecules are easily visualized under an ultraviolet lamp
when electrophoresed in the presence of the extrinsic Fluor ethidium
bromide.
• Ethidium bromide is a known mutagen or carcinogenic and should be
handled as a hazardous chemical - wear gloves while handling
• To prepare gel, agarose powder is mixed with electrophoresis buffer to the
desired concentration, and heated in a microwave oven to melt it. Ethidium
bromide is added to the gel (final concentration 0.5 ug/ml) to facilitate
visualization of DNA after electrophoresis.

16. • After cooling the solution to about 60℃ it is poured into a casting tray containing a
sample comb and allowed to solidify at room temperature.
• Samples containing DNA mixed with loading buffer are then pipetted into the
sample wells, the lid and power leads are placed on the apparatus and a current is
applied run the gel for 30-45 mins at 100-150V. The current flow can be confirmed
by observing bubbles coming off the electrodes
• DNA will migrate towards the positive electrode, which is usually colored red, in
view of its negative charge and separated based on their isoelectric points.

17. Technique combining ideas of isoelectric points and electric fields. It
gives good separation with a high resolution compared to any other
method

18. 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.

19. 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.