Ppt presentation on Isoelectric focusing by asif iqbal
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This document presents information on isoelectric focusing (IEF), which is an electrophoretic technique that separates proteins according to differences in their isoelectric point (pI). IEF requires a solid support like agarose or polyacrylamide gel containing a pH gradient, across which a voltage is applied. Each protein migrates within the gel until it reaches the point where it has no net charge, its pI. IEF provides high resolution separation and is useful for analyzing proteins. It works based on the theory that each protein has a unique pI that depends on its amino acid composition.
Ppt presentation on Isoelectric focusing by asif iqbal
- 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
- 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
Editor's Notes
- #3: 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.
- #4: 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)
- #19: mixture of specially designed amphoteric substances, so-called carrier ampholytes.