Ppt presentation on Ppt presentation on Isoelectric focusing Isoelectric focusing

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

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.

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)

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

Required for Isoelectric focusing

Sample Ampholytes Buffer Voltage Supporting medium Gel

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.

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.

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

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

Technique combining ideas of isoelectric points and electric fields.

It gives good separation with a high resolution compared to any other method.

IEFIEF

Amino acid

Polar no charge

Charged amino acids

Hydrophobic amino acids

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

A TYPICAL ISOELECTRIC A TYPICAL ISOELECTRIC FOCUSING GELFOCUSING GEL

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.

Isoelectric focusing (IEF)

cathode (-)

anode (+)

pH gradient

higher pH

lower pH

zero net charge

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.

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

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_purification.htm

Baskin E.F.; Bukshpan S; Zilberstein G V (2006). "pH-induced intracellular protein transport".

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