asif iqbal 03359107620 mphil microbiology

1. Ppt presentation onPpt presentation on
Isoelectric focusingIsoelectric focusing
By
Asif Iqbal Khattak
M.Phil Microbiology
2nd
semester
Reg no:9229
Abasyn university Peshawar

2. ISOELECTRIC FOCUSING
Electrophoretic method that separates
proteins according to differences in their
isoelectric point (pI).
Is ideal for separation of amphoteric
substances.
Separation is achieved by applying a
potential difference across a gel that contain
a pH gradient.

3. 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. 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.

5. 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

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

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

8. 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

9. IEFIEF

11. Polar no charge
Charged amino acids

12. Hydrophobic amino acids

13. 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

14. A TYPICAL ISOELECTRICA TYPICAL ISOELECTRIC
FOCUSING GELFOCUSING GEL

15. 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.

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

17. 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.

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

19. 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".

20. Thank you for listening
my presentation