This document presents a summary of isoelectric focusing, which is an electrophoretic technique that separates proteins based on differences in their isoelectric point (pI). Isoelectric focusing uses immobilized pH gradients to allow proteins to migrate through a gel until they reach the pH that corresponds to their individual pI, at which point they become neutrally charged and stop migrating. Key aspects covered include how proteins carry varying charges depending on pH, requirements for isoelectric focusing like ampholytes and voltage application, and uses like high-resolution protein analysis.

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Isoelectric focusingIsoelectric focusing
By
Asif Iqbal Khattak
M.Phil Microbiology
2nd
semester
Reg no:9229

2. ISOELECTRIC FOCUSING
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.
Is ideal for separation of amphoteric
substances.

3. 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.
Separates proteins by their isoelectric points
(pI) .
Each protein has own pI = pH at which the
protein has equal amount of positive and
negative charges
(the net charge is zero)

4. pI
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
negative.
pI is very specific for each protein

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

6. 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.

7. 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.

8. 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

9. IEF is well established as an excellent
technique for the analysis of proteins, such
as enzymes, hormones or other biologically
active proteins.

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

11. IEFIEF

12. Amino acid

14. Polar no charge
Charged amino acids

15. Hydrophobic amino acids

16. 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

17. A TYPICAL ISOELECTRICA TYPICAL ISOELECTRIC
FOCUSING GELFOCUSING GEL

18. 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.

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

20. 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.

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

22. References
Voet, D. Voet, J. G. Pratt. C. W. Fundamentals of
Biochemistry: Life at the Molecular Level. 3rd
edition. John Wiley and Sons. (2008)
http://www.science-tube.com/
http://www.zeitnews.org/
http://www.biochem.arizona.edu/classes/bioc462/
462a/NOTES/Protein_Properties/protein_purificat
ion.htm
Baskin E.F.; Bukshpan S; Zilberstein G V (2006). "pH-
induced intracellular protein transport".

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