The development of the technique of isoelectric focusing (IEF) represents a major advance in the field of high-resolution separations of proteins and other amphoteric macromolecules. IEF is an equilibrium method in which amphoteric molecules are segregated according to their isoelectric points (pl) in pH gradients. The pH gradients are formed by electrolysis of amphoteric buffer substances known as carrier ampholytes. When introduced into this system, other amphoteric molecules such as proteins migrate to pH zones that correspond to their respective pls where their net charge is zero. By counteracting back-diffusion with an appropriate electrical field the separated molecules can be concentrated into extremely sharp bands. The technique has now been refined to a level that permits the resolution of molecules whose pls differ by as little as 0.005 pH unit or less. This degree of resolution cannot normally be obtained by conventional electrophoretic or chromatographic procedures. In these latter procedures, specially adjusted conditions have to be devised for particular separations. While in contrast, IEF, by virtue of being an equilibrium method has a “built-in” resolution which usually allows one to separate in only one or two experiments all components with measurably different pl values. Further. because it is an equilibrium method, the system is self-correcting and therefore considerably less demanding in terms of experimental technique. IEF is particularly suitable for differentiating closely related molecules and provides a valuable criterion of homogeneity.

1. WELCOME
Department of Molecular Biology and Biotechnology
Advanced Centre for Plant Biotechnology, GKVK, UAS(B)

2. IEF of Proteins
Isoelectric focusing
Brahmesh Reddy B R
PAMB 2088

3. ISOELECTRIC FOCUSING (IEF)
Isoelectric focusing (IEF) is an electrophoretic method for
the separation of proteins, according to their isoelectric
points (pI), in a stabilized pH gradient.
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology
>What makes IEF powerful tool?

4. The combination of two features, at least, makes isoelectric
focusing appealing over other electrophoretic methods.
● First, IEF belongs to steady-state techniques ie. the equilibrium in
concentration distribution of the components under separation is
normally achieved at the end of process.
● Second, the isoelectric point (pI) represents a fundamental
property of a protein. It’s a unique physico-chemical constant for
a protein reflecting both its amino acid composition and the
conformation.
IEF, what makes it so powerful and
attractive?
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

5. Amino acid
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology
Carboxyl group
Amino group
Alpha Carbon

6. Amino acid - alanine
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

7. amino acids carry –COOH and -NH₂ groups in their side chains.
These groups contain both acidic and basic groups.
The carboxyl group is acidic and the amino group is basic
Zwitterion
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

8. Zwitterion
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology
The carboxyl group (–COOH) is acidic which donates a proton by
dissociation.
The amino group (-NH₂) is basic and can accept a proton.
When amino groups and carboxyl
groups are ionized, the amino
acids are known as zwitterion
>what is the condition required for a zwitterion formation?

9. Zwitterion
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology
The behaviour of a zwitterion

10. isoelectric point (pI)
The pH at which amino acids do not migrate towards the cathode
(or anode) in the presence of an electric field is referred to as the
“isoelectric point.”
● The isoelectric point (pI) is the pH at which a molecule has a
net charge zero.
● At this point, the molecule is electrically neutral and does
not migrate in an electric field.
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

11. Amino acid - alanine
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

12. Zwitterion of Glycine
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

13. Protein
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

14. Isoelectric Point (pl) of LYSOZYME
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology
3D render of lysozyme
pl of lysozyme
11.0

15. Isoelectric Point (pl) of INSULIN
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology
3D render of insulin
pl of insulin
5.4

16. Isoelectric Point (pl) of MYOGLOBIN
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology
3D render of Myoglobin
pl of myoglobin
7.0

17. Isoelectric Point (pl) of SERUM ALBUMIN
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology
3D render of Serum Albumin
pl of serum albumin
4.9

18. Isoelectric Point (pl) of RIBONUCLEASE
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology
3D render of RNase II
pl of ribonuclease
RNase II - 7.8

19. Why IEF?
When does isoelectric focussing come
into play?
What is the most common
method used to separate
proteins?
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

20. Why IEF?
When does isoelectric focussing come
into play?
What is the most common
method used to separate
proteins?
-SDS PAGE
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

21. Why IEF?
When does isoelectric focussing come
into play?
What is the most common
method used to separate
proteins?
-SDS PAGE
What happens in SDS PAGE
and what is the principle of
separation of proteins in
SDS PAGE?
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

22. What happens in SDS PAGE?
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

23. What happens in SDS PAGE?
● Only peptide chain remains
● With net negative charge-SDS
● Separation is based on
molecular weight only
The non-covalent bonds of proteins
are broken by SDS and β-
mercaptoethanol which denature
the 2° and 3° structure
Hence neither charge nor protein
structure play role in SDS-PAGE
separation
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

24. What if two proteins have same molecular weight?
Molecular weight
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

25. What if two proteins have same molecular weight?
Molecular weight
Isoelectric point
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

26. The solution - IEF
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

27. The solution - IEF
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

28. The solution - IEF
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

29. IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

30. IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

31. IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

32. IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology
We have completed separation in one dimension

33. IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

34. IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

35. IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

36. Methodology
Virtual Proteomics laboratory - IIT Bombay protocol
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

37. CASE STUDY - 1
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

38. CASE STUDY - 1
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

39. CASE STUDY - 1
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

40. CASE STUDY - 1
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

41. CASE STUDY - 1
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

42. CASE STUDY - 1
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

43. CASE STUDY - 1
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology
MATURE SEED

44. CASE STUDY - 1
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology
GREEN LEAF

45. CASE STUDY - 1
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology
FULLY OPENED FLOWER

46. CASE STUDY - 1
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology

47. thanks!!!
(23) The principle of 2D Gel Electrophoresis/and the isoelectric point - YouTube
(23) How Does Isoelectric Focusing Work (IEF EXPLAINED) - YouTube
IEF of
Proteins
Brahmesh
Reddy B R
MBB 504 (2+1) / Techniques in Molecular Biology - I
Department of Molecular Biology and Biotechnology