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Auxin (phytohormone) analysis

Auxin (phytohormone) analysis. Jakub Rolčík. O. R. O. N. H. A uxin. R = H, Amino acids. I ndol e-3-acetic acid (IAA). Amounts of phytohormones typically quantified in plant material. Phytohormones act at very low – “trace” - concentrations. Amounts expressed in mol :

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Auxin (phytohormone) analysis

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  1. Auxin (phytohormone) analysis Jakub Rolčík

  2. O R O N H Auxin R = H, Amino acids Indole-3-acetic acid (IAA)

  3. Amounts of phytohormones typically quantified in plant material Phytohormonesact at very low – “trace” - concentrations Amounts expressed in mol: 1 mmol = 0,001 mol 1 μmol = 0,000 001 mol 1 nmol = 0,000 000 001 mol 1 pmol = 0,000 000 000 001 mol 1 fmol = 0,000 000 000 000 001 mol 1 amol = 0,000 000 000 000 000 001 mol

  4. How to imagine “trace” concentration? Comparison with Gulliver in Brobdingnagu lags behind… Imagine the amount of phytohormone as an abscissa of 10 cm in length: 10 cm Then, what would be the length of the rest of the plant sample? Diameter of the Earth ! Aims of trace analysis: - To reduce the diameter of the Earth to a couple of mm - To measure the length corresponding to amount of auxin

  5. Imunization Myelomacells Fusion Splenocytes AH medium DOTBLOT Cloning DOTBLOT Cloning Nitrogen (l) in vitro in vivo Monoclonal Abs Monoclonal Abs Preparation of monoclonal antibodies • Polyclonal antibodies: Imunization of rabbits • Monoclonal antibodies Immunization of mice and hybridoma technology

  6. IAE & Derivatives of BAPHPLC-UV chromatograms – mouse serum processed by SPEand by combination of SPE & IAE

  7. 2.8e-3 2.6e-3 2.4e-3 2.2e-3 2.0e-3 1.8e-3 1.6e-3 1.4e-3 1.2e-3 1.0e-3 8.0e-4 AU 6.0e-4 4.0e-4 2.0e-4 -9.155e-11 -2.0e-4 -4.0e-4 -6.0e-4 -8.0e-4 -1.0e-3 -1.2e-3 -1.4e-3 Time 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 HPLC separation of auxins 6.02 IAM 19.92 IALeuMe 8.63 IAGlyMe 16.23 IAAMe 14.27 IAGluMe 11.48 IAAlaMe 17.45 IAValMe 12.60 IAAspMe 12.28 IAN 10.45 IEt 19.52 IPAMe 14.77 IMe 20.43 IAPheMe 18.27 IATrpMe 20.93 IBAMe

  8. UPLC (Ultra-Performance Liquid Chromatography) Novel kind of sorbent resistant to high pressure (1000 atm)

  9. UPLC – compared with HPLC – is significantly faster

  10. MS spectrum of compoundof M=288 g/mol

  11. Mass Spectrometry – Single Quadrupole

  12. Mass Spectrometry – Triple Quadrupole • Nabitý ion je selektován prvním kvadrupólem. • V druhém kvadrupólu (v kolizní cele) je ion bombardován atomy argonu. Vzniká směs menších iontů. • Třetím kvadrupólem je selektován ion typický pro analyt.

  13. Requirements - Deep understanding of phytohormone nature and chemistry - Experience and tradition - Patience and endurance (from 3 months to 3 years) - Good instrumentation (up to € 1,000,000) - Good people

  14. Understandable characteristics of current phytohormone analysis 10 mg – amount corresponding to a quarter of peeled sunflower seed – is sufficient for analysis • Outlook: Quantification of IAA in a single root tip • Examples: • fmol per root tip • fmol per mm

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