auxin (phytohormone) analysis
DESCRIPTION
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 : - PowerPoint PPT PresentationTRANSCRIPT
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Auxin (phytohormone) analysis
Jakub RolčíkJakub Rolčík
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AAuxinuxin
Indole-3-acetic acid (IAA)
N
OR
O
H
R = H, Amino acids
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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
PhytohormonesPhytohormonesact at very low – “trace” - concentrationsact at very low – “trace” - concentrations
Amounts of phytohormones typicallyquantified in plant material
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10 cm
How to imagine “trace” concentrationHow to imagine “trace” concentration? ? Comparison with Comparison with GulliverGulliver in in Brobdingnagu Brobdingnagu lags behindlags behind……
Imagine the amount of phytohormoneas an abscissa of 10 cm in length:
Then, what would be the length ofthe 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
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Imunization Myeloma cells
Splenocytes
Fusion
AH medium
DOTBLOT
Cloning
DOTBLOT
Cloning
in vitro in vivo
Monoclonal Abs Monoclonal Abs
Nitrogen (l)
Preparation of monoclonal antibodiesPreparation of monoclonal antibodies
- Polyclonal antibodies:
Imunization of rabbits
- Monoclonal antibodies
Immunization of mice and hybridoma technology
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2: D iode A rray 268 nm
8.91
250
Tim e
7 .00 8 .00 9 .00 10.00 11.000
100
%
2-O H -3-O C H3-BAPR
2: D iode A rray 268 nm
8.89
2-O H -3-O C H 3-B A P R
IAE & DIAE & Deeririvatives ofvatives of BAP BAP
HPLC-UV chromatogramHPLC-UV chromatogramss – – mousemouse serum serum processed by processed by SPESPE
aand by combination of nd by combination of SPE & IAESPE & IAE
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HPLC HPLC separation of auxinsseparation of auxins
Time5.00 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
AU
-1.4e-3
-1.2e-3
-1.0e-3
-8.0e-4
-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
1.6e-3
1.8e-3
2.0e-3
2.2e-3
2.4e-3
2.6e-3
2.8e-3
6.02
IAM
8.63
IAGlyMe
19.92
IALeuMe
16.23
IAAMe
14.27
IAGluMe11.48
IAAlaMe
10.45
IEt
12.60
IAAspMe12.28
IAN
14.77
IMe
17.45
IAValMe
19.52
IPAMe18.27
IATrpMe20.43
IAPheMe
20.93
IBAMe
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UPLC (UPLC (UUltra-ltra-PPerformance erformance LLiquid iquid CChromatographyhromatography))
Novel kind of sorbent resistant to high pressure (1000 atm)
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UPLC – UPLC – compared with compared with HPLC – HPLC – is significantly fasteris significantly faster
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18 StdsD / 5 (25 pmol) - FS - 10 mM
m/z100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
%
0
100
06032607 - 18SD (25 pmol) FS 919 (17.454) Cn (Cen,2, 80.00, Ht); Sm (Mn, 2x0.75); Cm (915:921-(911:914+922:929)) 1: Scan ES+ 3.67e6288.98
228.06212.12198.52116.81 149.03129.07 166.86 170.93
256.90232.65
286.93275.43
310.94
306.95
348.00
326.87328.37
356.89372.92 390.71 398.68
MS spectrum MS spectrum of compoundof compound of M=of M=288 g/mol288 g/mol
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Mass Spectrometry – Single QuadrupoleMass Spectrometry – Single Quadrupole
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Mass Spectrometry – Triple QuadrupoleMass 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.
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RequirementsRequirements
- 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
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Understandable characteristics of current Understandable characteristics of current phytohormone analysisphytohormone 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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