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1. Subject:- Laboratory Technique’s-II
Topic: Iso Electric Focusing
Group Members Names:
Sr# - Name’s - Roll No’s
1. Nooruddinadil - MLT/2K17/53
2. Sunil Kumar - MLT/2K17/72
3. Pershotam Sagar - MLT/2K17/55
4. Vikash - MLT/2K17/76
5. Heera Lal - MLT/2K17/26
6. Naresh Kumar - MLT/2K17/50
7. Doulat - MLT/2K17/20
8. Kanwer Lal - MLT/2K17/30

2. Context/Index
 Introduction to Iso Electric Focusing.
 Iso Electric Point.
 Requiment for Iso Electric Focusing.
I. Sample.
II. Ampholyte.
III. Buffer.
IV. Voltage.
V. Supporting medium.
VI. Gel. -
 Method of Iso Electric Focusing.

3. Introduction to Iso Electric Focusing
 IEF Stands For Iso Electric Focusing.
 IEF Stands for (iso=Same+ Electric=Charge + Focusing=Concentration)
so IEF=Same Charge Concentration
 IEF separates amphoteric compounds, such as proteins, with increased resolution in a medium possessing a
stable pH gradient
 Electrophoretic method that separates proteins according to differences in their isoelectric point (pI).
 Electrophoresis is the migration of charged molecules, particles or ion in a liquid medium under the
influence of an electric field.
 A procedure to determine the isoelectric point (PI) of proteins thus, a mixture of proteins can be
electrophorised 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
 Is ideal for separation of amphoteric substances.

4. Introduction to Iso Electric Focusing(Continued)…
 Separation 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.
 Each protein has own pI = pH at which the protein has equal amount of positive and negative
charges
(the net charge is zero)
 It gives good separation with a high resolution compared to any other method
 Resolution depends on
I. The pH gradient,
II. The thickness of the gel
III. Time of electrophoresis,
IV. The applied voltage,
V. Diffusion of the protein into the gel.

5. Introduction to Iso Electric Focusing(Continued)…
 Isoelectric focusing (IEF), also known as electrofocusing, is a
technique for separating different molecules by differences in
their isoelectric point (pI)
 IEF is performed into immobilized pH gradient (IPG) gels
 The PH gradient is formed in Polyacrylamide or agarose gel by
adding ampholytes solution
 IEF is well established as an excellent technique for the analysis of
proteins, such as enzymes, hormones or other biologically active
proteins.

6. Iso Electricpoint = pl
 Isoelectric focusing uses the theory of protein pI
 pI is the pH at which a given protein has a neutral overall charge
 The pI is dependent on which type of residues are present and how many.
 Bases make proteins positive and acids make negative.
 pI is very specific for each protein
 The point where the protein has a net charge of zero (that is, the sum of the negative and positive
charges equal to zero)
 Reached only when the pH = pI, as shown in the previous diagrams

7. Iso Electricpoint = pl (Contined….)
 The isoelectric point (pI, pH(I), IEP), is the pH at which a particular molecule carries no net electrical
charge
 Every amino acid has its own specific isoelectric point.
 Since proteins are made of amino acids, all proteins have their own pI
 Pre-albumin: pI ~ pH 4.7
 Albumin: pI ~ pH 4.9
 Gamma globulins: pI ~ pH 7.3
 A protein that is in a pH region below its isoelectric point (pI) will be positively charged and so will
migrate towards the cathode and vice versa
 When a protein is placed in a medium with a pH gradient and subjected to an electric field, it will
initially move toward the electrode with the opposite charge.
 During migration through the pH gradient, the protein will either pick up or lose protons.
 Above its isoelectric point, a protein has a net negative charge and migrates toward the anode
in an electrical field.
 Below its isoelectric point, the protein is positive and migrates toward the cathode.

8. ISOELECTRIC FOCSING
PRINCIPLE:
 All proteins have an isoelectric point=pH .
 When 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.
 Protein migrate into the point where its net charge is zero – isoelectric pH.
 Protein is positively charged in solutions at pH below its PI and will migrate towards the cathode.
 Protein is negatively charged in solution at pH above its PI will migrate towards the anode.
 They will be in the Zwitter ion form with no net charge so the further movement will cease.
 Ampholytes (amphoteric electrolytes)- low molecular mass (600-900D) ooligomers with aliphatic amino and carboxylic acid
groups with a range of isoelectric points. Ampholytes help maintain the pH gradiennt in the presence of high voltage.
 Can also use gels with immobilized pH gradients - made of acrylamide derivatives that are covalently linked to
ampholytes.

9. Required for Isoelectric focusing
Sample
Ampholytes
Buffer
Voltage
Supporting medium
Gel

10. 1.SAMPLE
 SAMPLE TYPES
1. PROTEIN
2. AMINO ACID
3. DNA
4. RNA

11. 2. AMPHOLYTE
 Ampholytes are complex mixtures of synthetic polyamino-polycarboxylic acids
 Types of Ampholyte
1. Synthetic carrier ampholyte’s.
2. Natural Occuring ampholyte’s.
 Commercially available ampholytes are-
1. BIO-LYTE
2. PHARMALYTE

12. Ampholyte (Continued….)
Synthetic carrier ampholyte v.s. natural
occurring ampholyte
 Synthetic carrier ampholyte:
 High buffering capacity and solubility at the pI.
 Good and regular electric conductivity Near 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.

13. 3.Buffer
Definition
 A buffer is a solution containing either a weak acid and its salt or a weak base and its
salt, which is resistant to changes in pH. In other words, a buffer is an aqueous
solution of either a weak acid and its conjugate base or a weak base and its conjugate
acid.
 Buffers are used to maintain a stable pH in a solution, as they can neutralize small quantities of
additional acid of base.
 For a given buffer solution, there is a working pH range and a set amount of acid or base that
can be neutralized before the pH will change. The amount of acid or base that can be added to a
buffer before changing its pH is called its buffer capacity.
 Types of Buffer
There are two general types of buffer
1. Acidic buffer
2. Basic buffer

14. 5.Supporting medium
SUPPORTING MEDIUM USED IN IEF
1. Filter Paper
2. Cellulose Acetate Membrane
3. Agrose
4. Polyacrylamide

15. 6. Gel
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 Used in IEF Method:
1. Agarose gel.
2. Polyacrylamide gel.

16. 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.

17. 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

18. METHOD
• pH gradient is established in gel by addition of ampholytes which increases the pH from anode to cathode.
• A protein mixture is placed in a well on the gel.
• With an applied electric field, proteins enter the gel migrates until each reaches its pH equivalent to its (PI).
• Each species of proteins is thereby focussed into a narrow band about its PI.
• The Anode of the column is connected to a reservoir containing an acidic solution like phosphoric acid and Cathode is
connected to a reservoir containing alkaline solution like sodium hydroxide.
• On opening the two reservoir valves the two solutions are allowed to diffuse into the column from their respective ends ,
setting up a PH gradient between the acidic anode and the alkaline cathode.
• The valves are then closed and the current is switched on , causing the carrier ampholytes to migrate until they reach the
PH regions where they have no net charge. They will then remain stationary at these points.

19. 19

20.  Technique combining ideas of isoelectric points and electric fields.
 It gives good separation with a high resolution compared to any other method.
METHOD (CONTINUE…….)

21. Iso Electric Point.

22. Amino acid
AMINO ACID TYPES ACCORDING TO ITS PH

23. How to Isoelectrofocus
 Establish a pH gradient
 Establish a voltage (> 1000 V)
 Stain your macromolecule (usually protein)
 Go do something while proteins migrates through the pH
gradient

24. A TYPICAL ISOELECTRIC FOCUSING GEL

25. Isoelectric focusing (IEF)
cathode (-)
anode (+)
pH gradient
higher pH
lower pH
zero net charge

26. What Happens
Proteins stop exactly at pH=pI and the stained proteins are very visible.

27. Isoelectric Focusing Methods (continued…)
 The overall charge of the protein is determined by the number of acidic
and basic amino acids in its basic structure.
 Because of their amphoteric nature, amino acids can express a net
positive charge, a net negative charge or a net charge of zero.

28. Isoelectric Focusing Methods (continued…)
 pH of the buffer (reagent) determines the charge of the molecule
 Net charge of molecule determines migration direction in electrical field