Chromatofocusing

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chromatofocusing chromatography

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Chromatofocusing

  1. 1. by Amlan Barai 13I300002 Department of Bio-Science & Bio-Engineering Indian Institute of Technology Bombay
  2. 2. pH and Protein pH=-log[H+] So, A protein at pH3 have more [H+] in its environment than a protein at pH9
  3. 3. pKa • pKa is the pH at which any group donates half of its ionisable proton pKa=-logKa • pKa tells us how acidic (or not) a given hydrogen atom in a molecule is. The stronger the acid, the lower its pKa; the stronger the base, the higher its pKa
  4. 4. pH pH 2.34 1 Amlan Barai, 13i300002, BSBE, IIT Bombay
  5. 5. pH 9.69 6 Amlan Barai, 13i300002, BSBE, IIT Bombay
  6. 6. Increasing the at pKa1 =2.34 pH Amlan Barai, 13i300002, BSBE, IIT Bombay
  7. 7. Decreasing the at pKa2 =9.69 pH Amlan Barai, 13i300002, BSBE, IIT Bombay
  8. 8. The Midpoint : pI or Isoelectric Point Amlan Barai, 13i300002, BSBE, IIT Bombay
  9. 9. So pI or Isoelectric Point is simply the pH at which the net charge is Zero.
  10. 10. Calculation of pI:the pKa & pI of Amino Acids Table: Modified from Lehninger Principle of Biochemistry, 5th ed. P-73.
  11. 11. pI of Protein above pI: -ve Below pI:+ve
  12. 12. So the Bottom line • Proteins and Amino Acids are Amphoteric molecules with positively and negatively charged groups, where their dissociation depends on the H+ ion concentration of the surrounding environment. • Molecules at pH above its pI have net –ve charge & Below its pI have net +ve charge.
  13. 13. Chromatofocusing • Separating on the basis of pI (Isoelectric Point) • First Discovered in 1978 by Sluyterman And His Colleagues
  14. 14. above pI: -ve Below pI:+ve Ref: http://macromol.sbcs.qmul.ac.uk/oldsite/expertise/CF3.jpg
  15. 15. The Elution profile of two Proteins above pI: -ve Below pI:+ve 9 8 Ref(modified from): Chromatofocusing- Douglas D Frey,Chittoor R Narahari, Ronald C Bates, Encyclopedia Of Life Sciences /&2001 Nature Publishing Group / www.els.ne
  16. 16. Focusing in chromato“focusing” Amlan Barai, 13I300002, BSBE, IIT Bombay. pH<8 pH=8(=pI) pH>8 + - pH<8 pH=8(=pI) pH>8 pH<8 pH=8(=pI) pH>8 + - pH<8 pH=8(=pI) pH>8 - Elution with Gradually increasing pH + + + + + + + + + + + above pI: -ve Below pI:+ve
  17. 17. Focusing in chromato“focusing” above pI: -ve Below pI:+ve
  18. 18. Chromatofocusing: The Assembly
  19. 19. The pH Gradient The self generated gradient 5 pH5 pH9
  20. 20. Buffers used for chromatofocusing Polybuffer 74 • Polybuffer 74 forms a linear pH gradients from pH 7 to 4. • Use Polybuffer 74 for any pH gradient between 7 and 4. • Developed for chromatofocusing, form linear pH gradients. • Mixtures of selected amphoteric buffering substances of different pI and pKa values. • Resolves pI differences of 0.04 pH units. Ref:GE Healthcare(http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences/products/AlternativeProductStructure_17391/17071201) & Amersham Pharmacia Biotechnology.
  21. 21. Buffers used for chromatofocusing Polybuffer 96 • Polybuffer 96 forms a linear pH gradients from pH 9 to 6. • Developed for chromatofocusing, form linear pH gradients. • Mixtures of selected amphoteric buffering substances of different pI and pKa values. • Resolves pI differences of 0.04 pH units. • Use Polybuffer 96 for pH gradients that should begin above pH 7. Ref:GE Healthcare(http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences/products/AlternativeProductStructure_17391/17071201) & Amersham Pharmacia Biotechnology.
  22. 22. Beads for chromatofocusing PBE 94 (Polybuffer exchange94) • PBE 94 is a bead-formed exchanger gel (Sepharose®). Charged groups has coupled with them via ether linkage. • It has an even capacity over a wide pH range. • It is developed specifically for chromatofocusing with Polybuffer™ • Highly stable can even function in presence of 8M Urea and at 120°C. Ref:GE Healthcare(http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences/products/AlternativeProductStructure_17391/17071201) & Amersham Pharmacia Biotechnology.
  23. 23. Application • Separating proteins according to isoelectric point (pI) • It is a powerful method for high resolution, since it can resolve very small differences in pI (down to 0.02-0.05 pH units) and thus separate very similar proteins. • Used for analytical separations.
  24. 24. Applied for • Separation of two isoforms of the proteinb2-macroglobulin that differ by a single amino acid residue(Odani H, Oyama R, Titani K, Ogawa H and Saito A (1990)Biochemical and Biophysical Research Communications) • Purification and concentration of proteins produced by Haemophilus influenzaefor use in proteome analysis (Fountoulakis M, Langen H, Gray C and Takacs B (1998)Journal of Chromatography A806: 279–291) • Preparative-scale separation and purification of the peptides thymosinb4 and thymosinb9 from bovine tissue (Roboti A, Livaniou E, Evangelatos GPet al. (1994)Journal of Chromatography A662:27–34) • Separation of cortisol–bovine serum albumin conjugates (Giraudi G and Baggiani C (1990)Analyst115: 1531–1534)
  25. 25. Reference • Protein Liquid Chromatography-edited by Michael Kastner Chapter7-Chromatofocusing:Richard Lukacin and Wolfgan R. Deppert (http://books.google.co.in/books?id=3WhftkdNpxYC&dq=chromatofocusing+principle) • Chromatofocusing- Douglas D Frey,Chittoor R Narahari, Ronald C Bates, Encyclopedia Of Life Sciences /&2001 Nature Publishing Group / www.els.ne • Amersham Pharmacia Biotechnology (1987)Chromatofocusing with Polybuffer and PBE. Uppsala: Amersham. • Chromatofocusing :L . A . Ae . Sluyterman and J . Wijdenes Isoelectric Focusing On Ion-exchange Columns Journal of Chromatography, 150 (1978) 31-44Q Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands • GEHealthcare(http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSci ences/products/AlternativeProductStructure_17391/17071201)

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