Ppt presentation on 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.

Electrophoresis is a method whereby charged molecules in solution, chiefly proteins and nucleic acids, migrate in response to an electrical field. Proteins are amphoteric compounds, that is, they contain both acidic and basic residues. Potential difference is the energy supplied by a unit positive charge as it moves from a point of higher potential to a point of lower potential.*

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)

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)*

pIIsoelectric focusing uses the theory of protein pIpI is the pH at which a given protein has a neutral overall chargeThe 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 focusingSampleAmpholytesBufferVoltageSupporting mediumGel

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 GELA 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 40C.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.

*

IEF

Amino acid

Polar no chargeCharged amino acids

Hydrophobic amino acids

How to IsoelectrofocusEstablish a pH gradientEstablish a voltage (> 1000 V) Stain your macromolecule (usually protein)Go do something while proteins migrates through the pH gradient

A TYPICAL ISOELECTRIC FOCUSING 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 gradienthigher pH

lower pHzero 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 HappensProteins stop exactly at pH=pI and the stained proteins are very visible .

mixture of specially designed amphoteric substances, so-called carrier ampholytes. *

ReferencesVoet, 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".

*

Thank you for listening my presentation

*

Electrophoresis is a method whereby charged molecules in solution, chiefly proteins and nucleic acids, migrate in response to an electrical field. Proteins are amphoteric compounds, that is, they contain both acidic and basic residues. Potential difference is the energy supplied by a unit positive charge as it moves from a point of higher potential to a point of lower potential.*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)*

*

*

*

*mixture of specially designed amphoteric substances, so-called carrier ampholytes. **