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Isoelectric focusing
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 focusing
- 1. Ppt presentation onPptpresentation on Isoelectric focusingIsoelectric focusing By Asif Iqbal Khattak M.Phil Microbiology 2nd semester Reg no:9229
- 2. ISOELECTRIC FOCUSING Electrophoretic methodthat 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 achievedby 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 usesthe 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 Isoelectricfocusing Sample Ampholytes Buffer Voltage Supporting medium Gel
- 6. What is agel? 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 Ahighly 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 toseparate 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 wellestablished as an excellent technique for the analysis of proteins, such as enzymes, hormones or other biologically active proteins.
- 10. Technique combining ideasof isoelectric points and electric fields. It gives good separation with a high resolution compared to any other method.
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- 14. Polar no charge Chargedamino acids
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- 16. How to Isoelectrofocus Establisha 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 ISOELECTRICATYPICAL ISOELECTRIC FOCUSING GELFOCUSING GEL
- 18. When a proteinis 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 isoelectricpoint, 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 Proteinsstop 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".
- 23. Thank you forlistening 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)
- #22 mixture of specially designed amphoteric substances, so-called carrier ampholytes.